Mechanistic Basis of Electron Transfer to Cytochromes P450 by Natural Redox Partners and Artificial Donor Constructs

Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 851)

Abstract

Cytochromes P450 (P450s) are hemoproteins catalyzing oxidative biotransformation of a vast array of natural and xenobiotic compounds. Reducing equivalents required for dioxygen cleavage and substrate hydroxylation originate from different redox partners including diflavin reductases, flavodoxins, ferredoxins and phthalate dioxygenase reductase (PDR)-type proteins. Accordingly, circumstantial analysis of structural and physicochemical features governing donor-acceptor recognition and electron transfer poses an intriguing challenge. Thus, conformational flexibility reflected by togging between closed and open states of solvent exposed patches on the redox components was shown to be instrumental to steered electron transmission. Here, the membrane-interactive tails of the P450 enzymes and donor proteins were recognized to be crucial to proper orientation toward each other of surface sites on the redox modules steering functional coupling. Also, mobile electron shuttling may come into play. While charge-pairing mechanisms are of primary importance in attraction and complexation of the redox partners, hydrophobic and van der Waals cohesion forces play a minor role in docking events. Due to catalytic plasticity of P450 enzymes, there is considerable promise in biotechnological applications. Here, deeper insight into the mechanistic basis of the redox machinery will permit optimization of redox processes via directed evolution and DNA shuffling. Thus, creation of hybrid systems by fusion of the modified heme domain of P450s with proteinaceous electron carriers helps obviate the tedious reconstitution procedure and induces novel activities. Also, P450-based amperometric biosensors may open new vistas in pharmaceutical and clinical implementation and environmental monitoring.

Keywords

P450 Redox machinery Key determinants Genetic engineering Biotechnological exploitation 

10.1 Introduction

Cytochrome P450 (CYP or P450) enzymes, occurring in organisms from all domains of life [1, 2, 3, 4, 5], represent a superfamily of ever-growing b-type heme-thiolate proteins [6]. The metalloenzymes are of major importance in both the biosynthesis of endogenous compounds [7, 8] and oxidative clearance of a vast array of drugs, toxins and environmental pollutants characterized by high structural diversity [9, 10]. These processes require the consecutive delivery of two electrons to the ferric P450 catalysts to convert the unreactive atmospheric dioxygen via a generally accepted O-O bond activation cycle to a high-valent iron-oxo species capable of attacking C-H entities and heteroatoms in substrate molecules [11, 12]. Apart from this consensus mechanism, recent data spark particular interest in a “multi-oxidant” concept, providing a rationale for the striking catalytic diversification of P450s [13, 14, 15].

Although it would appear that the plethora of CYP genes evolved from a common ancestor [16], there exist variations in the nature of the intermediate carrier systems bridging NAD(P)H-derived reducing equivalents to specific terminal P450 acceptors [17]. Thus, in class I P450s comprising bacterial and eukaryotic mitochondrial hemoproteins, a flavin-containing ferredoxin reductase (FdR) usually operates in conjunction with an [Fe2-S2] cluster-bearing ferredoxin (Fdx) to shuttle electrons from the reduced cofactor to the heme iron [18]. Noteworthy, in the CYP107H1- and CYP176A1-dependent microbial electron transport chains, unusual FMN-carrying flavodoxins act as functional substitutes for ferredoxins [19, 20, 21, 22, 23, 24]. In the class II monooxygenase apparatus comprising microsomal P450 proteins, FAD/FMN prosthetic components in the structure of NADPH-cytochrome P450 oxidoreductase (POR) foster swift electron delivery to the various candidates; here, the NADH-driven cytochrome b5 (b5)/b5 oxidoreductase pair can serve as an alternate redox partner [18]. On the other hand, the unique CYP55A1 enzyme, promoting reductive conversion of nitric oxide to the gaseous nitrous oxide, utilizes NADH as a direct electron supplier without the need for any auxiliary mediator [25]. With other P450s such as human CYP2S1 or bacterial CYP152A/B, the typical O2/2e/2H+ proteinaceous systems fail to stimulate catalytic activity, while utilization of H2O2 or fatty acid hydroperoxides permits efficient substrate turnover via the peroxygenase main route based on homolytic peroxy O-O bond scission [26, 27, 28]. Similarly, biocatalysts such as CYP5A or CYP74, bringing about rearrangement of endoperoxides and hydroperoxides, respectively, require neither oxygen nor an NAD(P)H-type electron source [29, 30].

As can be readily seen, the pronounced P450-dependent specification of the redox machinery creates the challenging task of more detailed analysis of the structural and functional characteristics of the diverse electron transfer entities to improve our understanding of the observed electrochemical phenomena. In this respect, molecular modeling of composite 3D P450 constructs on the basis of the crystal structure, chemical modification and genetic engineering of a broad spectrum of hemoproteins provided an appreciable picture of both the overall topology of key determinants dictating donor docking/orientation and the nature of the driving forces supporting these events [31, 32]; this also helped assess the redox dynamics of the systems [33]. Circumstantial insight into these processes will be beneficial to the development of novel strategies serving to simplify transmission of reducing power, such as efficient installation of the peroxide shunt pathway to overcome the prohibitive costs for NAD(P)H as the constant electron donor or curtailing of the complex electron transfer conduits [34]; this will give an impetus to exploitation of more flexible P450s in biotechnological areas encompassing the production of fine chemicals, drug processing or degradation of environmental pollutants [35, 36]. The present chapter thus highlights significant breakthroughs in our knowledge about the mechanistic basis of donor/acceptor interactions in the functionally diversified domain of P450s, paving the way for innovative tailoring of versatile redox modules.

10.2 Mechanistic Principles of Electron Transport by Natural Redox Partners of P450s

10.2.1 NADPH-Cytochrome P450 Oxidoreductase (POR)

10.2.1.1 Evolutionary History

Microsomal POR represents a prototypic member of the fairly small family of diflavin redox proteins. The enzyme bears one molecule each of FAD and FMN as cofactors and favors electron transfer from NADPH to eukaryotic P450s or cytochrome c as the ultimate acceptors [37, 38]. Precursors of the 78-kDa POR proteins have been hypothesized to arise from ancestral fusion of genes encoding an FMN-binding bacterial flavodoxin and a plant-type FAD-complexed ferredoxin-NADP+ reductase. Subsequent evolutionary steps helped create an α-helical interdomain linker, allowing efficient functional coupling of the two flavins and an N-terminal membrane anchor region (Fig. 10.1) [39, 40, 41]. Flavodoxins as such operate in photosynthetic processes or participate in nitrate reduction as well as in methionine and biotin producing pathways [42]. Similarly, ferredoxin-NADP+ reductases display high functional plasticity in supporting auto- and heterotrophic reactions [43].
Fig. 10.1

Molecular evolution of NADPH-cytochrome P450 oxidoreductase (POR). The mammalian diflavin protein POR originates from ancestral fusion of the genes of flavodoxin and ferredoxin oxidoreductase with the subsequent creation of a flexible interdomain linker and a membrane anchor serving in proper orientation of the electron donor toward P450s (Data taken from Ref. [51])

Analysis of the genetic code for representative PORs from taxonomically diverse eukaryotic species mostly points at the involvement of a single gene in protein expression. Thus, the human gene, located on chromosome 7, contains 16 exons and has been found to be highly polymorphic [44, 45, 46]. Likewise, the rat gene carries 16 exons, 15 of which are coding exons. Organization of the latter strictly correlates with functional or structural domains [47]. Moreover, cytogenetic mapping of insect and fungal oxidoreductases suggests them to be single-copy products [48, 49]. Exception to this rule is given by the widespread polyploidy in plants, giving rise to gene duplication and divergence. In this respect, about 54 gene sequences encoding PORs derived from a total of 35 different plant species have as of now been identified, most of the paralogous enzymes at least partially complementing each other [50, 51]. Multiple-alignment studies revealed the majority of full-length POR proteins isolated from mammalian, insect, fungal and plant phyla to share 33–38 % amino acid sequence homology [49].

10.2.1.2 Electrochemical Features of Electron Transfer

The family of POR proteins mediates electron transfer in the NADPH→FAD→FMN→P450 redox system. Here, the flavin cofactors have a vital function in the step-down process from the obligatory two-electron donor NADPH to the one-electron acceptor P450. Using rabbit POR as a probe, flavins were shown to exist as one-electron reduced air-stable blue (neutral) semiquinones (FMN/FMNH, \( {E}_0^{\prime }=-110\kern0.24em \mathrm{mV} \) and FAD/FADH, \( {E}_0^{\prime }=-290\kern0.24em \mathrm{mV} \)) or two-electron fully reduced red (anionic) forms (FMNH/FMNH2, \( {E}_0^{\prime }=-270\kern0.24em \mathrm{mV} \) and FADH/FADH2, \( {E}_0^{\prime }=-365\kern0.24em \mathrm{mV} \)) equilibrating between these states [52, 53, 54, 55]. Noteworthy, no shift from the blue di-semiquinone (FMNH, FADH) toward the red species is observed upon increasing the pH of the reaction media [56]. However, the lipid bilayer of membrane-tethered POR was found to impact the redox potential of the FMN/FAD prosthetic groups: application of anionic phospholipids was shown to drive the E0 for the red forms of both cofactors to more negative values, favoring electron transfer to P450s [57]. Similarly, structural aberrations in the flavin-binding domains of reductases from different taxa may dramatically affect the redox parameters and abilities to support the catalytic activity of P450s. For example, the redox potential of the FMNH/FMNH2 pair in yeast POR was shown to be more positive than that for the blue couple. This behavior contrasts the situation in the rat and plant POR species and is similar to the inversion observed with the reductase moiety of the bacterial flavocytochrome CYP102A1 [58, 59].

Elucidation of the precise pathway of flavin-driven redox cycling during electron donation to heme catalytic centers has been fuelled by techniques such as deflavination and reconstitution [60] or dissection of PORs into their component domains [61], permitting more detailed studies on the kinetic and thermodynamic properties of the enzymes. Thus, triggering of the cycle is thought to be brought about by the stable, 635 nm-absorbing FAD-FMNH semiquinone potentially generated during a priming reaction [62]. Upon hydride transfer from NADPH (\( {E}_0^{\prime }=-320\kern0.24em \mathrm{mV} \)) to the latter species, interflavin electron flow proceeds from FADH-FMNH to yield FADH-FMNH. At high molar excess of NADPH, this process is reversible [63]. However, under in vivo conditions, the FMNH entity acts as the major one-electron supplier to the ferric heme iron of P450s, thereby returning to the resting semiquinone form in the FADH-FMNH duo [52, 64]. Electron cycling between the essentially equipotential members of this redox couple to regenerate FMNH seems fairly unfavorable and, indeed, occurs as a single-exponential process at a modest rate of 55 s−1, even dropping to a value of 11 s−1 when dithionite substitutes for NADPH as the reductant. This suggests cofactor binding to play a pivotal role in regulating internal electron flux [65]. Fully reduced FMN released in the gated electron transfer event serves in P450 reduction via a one-electron step. In summation, microsomal PORs usually cycle in a 1-3-2-1 sequence, denoting the total number of electrons carried by the flavins (Fig. 10.2) [64, 66]. Opposite to this, the microbial CYP102A1 fusion protein undergoes a reduction cycle of 0-2-1-0 lacking any priming reaction [67].
Fig. 10.2

POR-supported intra- and intermolecular electron conduction to P450s. Hydride transfer from NADPH to the stable semiquinone (a) elicits sequential formation of the fully reduced (b, c) intermediates to enable one-electron supply to ferric P450 associated with release of a resting semiquinone duo (d). Electron swapping between the latter redox couple regenerates a fully reduced cofactor (e) again permitting electron donation to P450s. The mammalian oxidoreductase thus cycles between the 1- and 3-electron reduced states

10.2.1.3 Structural Elements Governing Intramolecular Electron Transfer

A drastic step forward in the study of functional domains in POR proteins was made by comparison of the full-length amino acid sequences derived from a broad spectrum of species to unveil highly conserved signature motifs amenable to circumstantial analysis by genetic engineering [49, 50]. Moreover, availability of crystallographic data for human, rat and yeast PORs [68, 69, 70] enabled three-dimensional modeling of critical enzyme structures [71, 72]. Thus, investigation of the N-terminal α-helical signal anchor segments of mammalian oxidoreductases disclosed the carboxy termini to be located on the cytoplasmic side of the endoplasmic reticulum, with the first 55–56 amino acid residues being sufficient for stable membrane insertion/retention, proper orientation and maintenance of catalytic efficiency [73, 74, 75].

Despite the fairly low overall sequence identity (33–43 %) of POR enzymes from various organisms [49, 50], the FMN-binding domains exhibit a high degree of conservation [76]. Chain tracing in the scaffolds of human and rat proteins revealed an α-β-α architecture composed of a five-stranded parallel β-sheet in the core fold flanked by a variable number of α-helices, with the FMN cofactor positioned at the tip of the C-terminal side of the β-sheet [69, 71, 77]. Using the human enzyme (hPOR; NCBI reference sequence NP_000932.3) as a template, molecular docking and site-directed mutagenesis experiments suggested a set of residues such as Q90, T91, T142, H183 and N185 to be involved in FMN fixation, though Y143 and Y181 obviously act as key players [76, 78, 79]. The aromatic side chains of the two tyrosines, sitting on the re- and si-face, respectively, of the isoalloxazine ring, clasp the FMN unit at nearly the same distance of 3.5 Å [71, 77]. Both positions are conserved in the rat homolog [69] sharing 94 % sequence identity with the human counterpart [79], and Y→D exchange in the rodent protein was shown to indeed block efficient electron transfer [80]. Moreover, replacement in the human catalyst of F184, lying close to the pyrimidine tail of the cofactor, with leucine or glutamine caused a 40- to 50-fold increase in the Kd value for FMN association. This was interpreted to reflect a vital role of F184 in stabilization of the electron carrier [78]. Strikingly, L86 and L219, deeply buried in two hydrophobic cores of the FMN domain of POR from Anopheles minimus, aligns with F86 and F219 in the human analog. Experimental introduction into the insect enzyme of phenylalanine residues in place of the leucines proved to be beneficial to FMN docking and protein folding [75]. Of note, X-ray crystallography of oxidoreductase from the yeast Saccharomyces cerevisiae helped discover a second FMN-binding region at the interface of the linker and standard cofactor-bearing domain. The novel site displays low conservation throughout the gene family, with only two residues, namely, T71 and D187 corresponding to T93 and D211 in hPOR, being invariant [70]. It has been hypothesized that a single FMN molecule shuttles between the structural doublet associated with semiquinone transition from the neutral to the anionic state [70].

A fragment spanning about 40 amino acid residues of predominantly polar character bridges the gap between the FMN/FAD-harboring loci. This linker is speculated to serve in proper orientation of the flavins, the isoalloxazine rings of which make an angle of about 150° to each other and reside at a minimum distance of 3.5 Å [41, 69, 79]. In fact, mutations in the short random-coil hinge, preceding the FAD-connecting unit composed of residues G235 to R246 in the hPOR structure, induce drastic rearrangement of the FMN/FAD topology impacting intramolecular electron transfer [81]. Crystallographic analysis of the FAD-docking region in the rat protein showed the isoalloxazine entity to be hosted at the boundary between the cofactor- and NADPH-binding site, with the remainder of the molecule extending to the interface between the FAD-binding pocket and the connecting domain [69]. Site-directed mutagenesis was used to verify the functional importance of the various determinants. This demonstrated Y456 to make van der Waals contact with the si-side of the FAD isoalloxazine structure and to hydrogen-bond to the ribityl 4′-hydroxyl, while amino groups in the side chains of R454 (equivalent to R457 in hPOR), G488 and T491 stabilize the negatively charged pyrophosphate. Moreover, the aromatic nucleus of Y478 stacks on one side of the adenine moiety [68, 69, 82]. Interestingly, the interplay of the S457/D675/C630 triad may have a dual role in the control of the flavin redox potential and stabilization of the transition state to facilitate hydride transfer [83, 84].

A set of homologous residues lining the NADPH-binding cavity, constituted of alternating α-helices and β-strands, operate in fixation and orientation of the cofactor in a bipartite mode. Thus, highly conserved amino acids encompassing C566, S596, R597, K602 and Y604 (rat POR numbering) make up a specific motif attracting the 2′-phosphate of NADPH via H-bonding or salt-bridging, such as to cause discrimination against NADH [79, 85, 86, 87]. In accord with this, introduction of hydrophobic elements in place of the positively charged arginine and lysine residues at positions 597 and 602 to create the triple mutant R597M/K602W/W677A resulted in a 170-fold increase in the apparent binding affinity for NADH compared to the wild-type enzyme, paralleled by an IC50 value for inhibition by NADP+ that was 50-times higher than that of the parent enzyme [88]. Similarly, simple W676A exchange in hPOR allowed the NADH-dependent reductive potency to become equivalent to that of the NADPH-driven event [87]. On the other hand, this manipulation was found to compromise NADPH-promoted reduction beyond the two-electron level owing to slow release of NADP+ from the active site upon first hydride transfer, suggestive of a vital function of the C-terminal tryptophan in electrochemical processes [89]. The mechanistic basis of such an action relies on the assumption that the π-stacking indole of W677, building a lid above the re-face of the FAD isoalloxazine, moves away to permit direct contact of the flavin moiety with the nicotinamide ring of NADPH required for efficient hydride transfer [69, 72]. Here, local movement of the short G631-N635 loop may be beneficial to NADPH/NADP+ binding/release [90]. An overall diagram of the POR polypeptide fold disclosing the topology of the diverse cofactor-binding domains is presented in Fig. 10.3.
Fig. 10.3

Ribbon diagram illustrating the overall polypeptide fold and topology of POR. The FMN-binding domain is given in blue, while the NADP(H)- and FAD-docking sites are presented in green. The connecting interdomain fragment is depicted in red. Cofactors are presented in the ball and stick mode (PDB ID: 1AMO) (Reproduced from Ref. [69])

It should be pointed out that POR enzymes are highly polymorphic proteins. Currently, about 48 missense mutations have been identified in the human reductase (www.cypalleles.ki.se/por.htm), part of which overlap with conserved residues critical for FMN/FAD/NADPH fixation [45, 79, 91]. Examples of this are found in the most important variants T142A, Y181D, R457H, Y459H, V492E, C569Y, R600W and Y607C [45, 79]. These amino acid substitutions have been recognized to be deleterious to electron transfer and, consequently, cause disordered steroidogenesis along with skeletal malformations when the allelic hPORs are to serve as obligatory donors to CYP17A1 and CYP19A1 [91, 92]. Moreover, the altered phenotypes may impact the P450-catalyzed metabolic biotransformation of both curative drugs/prodrugs and toxins [45, 46, 93]. Noteworthy, the Y181D-induced perturbation of electron flow/P450 activity has been reported to undergo restoration upon the addition of excess FMN to the assay media [76, 94]. In this case, the potential existence of a second FMN-binding site (see above) might permit the exogenous cofactor to act as a bypass.

10.2.1.4 Structural Features Steering Functional POR Docking to P450s

To bring about efficient electron shuttling from POR enzymes to P450s, a large-scale conformational rearrangement of the FMN domain is required. Available 3D structures of mammalian reductases display a closed conformation of the core region with the isoalloxazine ring of FMN being shielded by the FAD cofactor at a distance ranging from 4 to 5 Å [68, 69]. Hence, electron donation to P450s necessitates concerted movement of the domains leading to an open state associated with exposure of the FMN moiety to the solvent to enable contact with the hemoproteins. In this regard, the “closed-open” transition, as studied with free or membrane-anchored POR, was recognized to be conducted by the flexible hinge motif adjusting the distance between the FAD/FMN entities to 29–60 Å [81, 95, 96]. More detailed analysis by sophisticated spectroscopic techniques revealed the POR molecule to, indeed, toggle between a multiplicity of closed and open conformations in solution [97, 98]. Generally, opening is driven by flavin reduction, whereas closure predominates in the oxidized enzyme and is supported by NADPH binding to facilitate loading of reducing equivalents [99, 100]. These findings are in line with the “swinging” model of POR-mediated electron transfer from the nicotinamide coenzyme to the heme iron of P450s [101].

Based on the construction of model complexes between the redox partners, a docking area of ~870 Å2 was calculated to guide productive encounter of the solvent-exposed FMN domain with P450s [81]. This patch, located on the surface of the extended reductase molecule, bears an electronegative profile arising from accommodation of three clusters of putative salt-bridging residues encompassing E92, E93, D113, E115, E116, E142, D144, D147 and D208 (rat POR numbering), speculated to provide a rationale for snuggly fit of the electropositive proximal face of the different P450s obviously binding in a very similar fashion [69, 71, 81, 102, 103]. The negatively charged elements surrounding the FMN moiety were predicted to form a cleft allowing a minimal distance of ~12 Å between the cofactor and the heme group [81]. The hypothetical acidic contact sites were substantiated by genetic engineering: Mutation of D113, E115 and E116 to alanine disclosed the residues to stabilize the CYP2B1/POR adduct on the one hand and open new avenues to more efficient electron transfer to the hemoprotein partner on the other [103]. Moreover, replacement of hPOR amino acids corresponding to E142, D144 and D147 in the rat homolog with the less bulky polar serine or glycine substitutes was found to moderately impinge on the catalytic efficiency (kcat/Km) of CYP2D6, while D208N exchange caused a drastic fall in P450-dependent activities [71, 104]. Two thirds of the determinants examined display 60–90 % conservation across the multitude of taxonomically diverse reductase species, the rest being invariant [76].

10.2.2 Cytochrome b5

Cytochrome b5 (b5), occurring in a wide range of phyla, is a membrane-anchored amphipathic hemoprotein operating in concert with POR or NADH-cytochrome b5 oxidoreductase as electron donor to desaturating systems involved in fatty acid synthesis and plasmalogen-producing enzymes [105]. Of note, soluble forms of human b5 and NADH-cytochrome b5 oxidoreductase found in erythrocytes were shown to be capable of reducing methemoglobin [106, 107]; here, deletion of codon 298 in the gene of the flavoprotein component was detected to cause functional deficiency associated with hereditary methemoglobinemia [108]. Moreover, the ferrihemoglobin-coupled redox triad brought about O2-dependent substrate turnover in a monooxygenase-type reaction [109]. In parallel, a considerable number of P450s were recognized to have substrate-specific obligatory requirement for electron supply by b5 [110, 111].

10.2.2.1 Topology of the Membrane-Spanning and Heme-Binding Domains of Cytochrome b5

Two mammalian b5 isoforms were identified, one inserted into the endoplasmic reticulum and the other bound to the outer membrane of mitochondria. These proteins arise from different genes [112]. The hydrophobic membrane anchor of the microsomal homolog, functioning as a static retention signal, was shown to span the bilayer of the endoplasmic reticulum such that the carboxy-terminus extends to the lumen of the organelle [113, 114]. However, mutation of the C-terminal L124/M125/Y126 triad in rat b5 to alanine was found to induce location of the engineered hemoprotein in both the cytosol and microsomal membrane, suggestive of the existence of loosely- and firmly-integrated forms differing by the overall content of α-helical structure [115, 116]. In fact, the membrane-interactive tail of b5 has been detected to function as a stop-transfer sequence giving rise to inversion of protein orientation in the endoplasmic reticulum to permit versatile processing of nascent precytochrome b5 during topogenesis, resulting in final positioning of the integral electron carrier in the Nout-Cin mode [117]. Another triad of potential interest embedded in the 43-amino-acid membrane-binding domain of b5 refers to tryptophan residues at locations 108, 109 and 112. However, studies with the W108L/W112L double mutant failed to disclose any impact on electron transfer to CYP2B4 as a probe acceptor [118]. Finally, attention was drawn to P115, forming a 26° kink in a helix when occurring in the trans conformation. Surprisingly, P→A exchange resulted in normal insertion of the mutant into the membrane and a wild-type enzyme level of activity in a P450 test system [119].

Microsomal b5, being 60 % α-helical, is a fairly small polypeptide composed of 134 amino acid residues, with the cytosolic heme-containing region showing ~92 % sequence identity throughout the different mammalian isoforms [105, 120]. Availability of the crystal and solution structure of the protein permitted insight into the architecture of the heme-binding pocket. Thus, the prosthetic group was shown to reside in a hydrophobic crevice, the iron atom being coordinated to histidines at positions 39 and 63; the latter reactant has some exposure to solvent via a water channel [121, 122, 123]. Dependence of the heme-holding stability on the histidine axial ligation was confirmed by H39S/C mutations, also affecting the spin state of the heme iron [124]. Apart from steric factors, changes in hydrophobicity of the heme microenvironment may modulate the electrochemical properties of the hemoprotein [125]. In accord with this, V45H/E substitutions were found to shift the redox potential of the wild-type protein (\( {E}_0^{\prime }=-10\kern0.24em \mathrm{mV} \)) to values of +8 mV and −26 mV, respectively [126]. Similarly, manipulation of hydrophobicity by replacement of V61 with histidine revealed to influence interaction of the heme with its pocket, resulting in broadening of the latter; this moved E0 of the mutant by +21 mV [127]. Special interest focuses on the interplay of the F35/F58 duo, stabilizing heme docking through π-stacking overlap with the porphyrine macrocycle [128, 129]. Moreover, phenylalanine-35 is part of a hydrophobic patch of 350 Å2 on the surface of b5 [130] and member of a network that includes Y74 and the axial H39 being in direct van der Waals and electrostatic contact with the heme [131, 132]. Apart from this, the conserved F35 is pivotal to fine tuning of the redox potential: F35Y exchange was discerned to make E066 mV more negative compared to the parent protein [133]. Finally, P40, another component of the surface patch producing a sharp γ-bend in the polypeptide chain, is believed to significantly contribute to a fixed folding pattern of the heme pocket due to its rotational restriction [134]. It should be kept in mind that, in contrast to the crystalline state, b5 is heterogenous in solution due to the presence of two isomers, differing with respect to 180° rotation of the heme plane around the axis defined by α,γ-meso protons [135]. A diagram of key structural motifs in the b5 backbone chain is given in Fig. 10.4.
Fig. 10.4

Schematic structure of bovine cytochrome b5. The approximately cylindrical molecule (PDB ID: 1CYO) houses α-helices 2–5 (in red) clustering around the prosthetic heme group given in grey balls and sticks. A five-stranded β-sheet (in yellow) in the center of the amphiphilic polypeptide separates the heme-binding pocket from a more peripheral helical segments 1 and 6 (in violet) (Data taken from Ref. [122])

10.2.2.2 Interaction of Cytochrome b5 with Electron Donors

The microsomal FAD-containing NADH-cytochrome b5 oxidoreductase acts as a physiological electron donor to the ferric b5. Anaerobic photoreduction of the FAD moiety was observed to form the red anionic semiquinone being in equilibrium with the blue neutral species. The latter turned out to be the primary intermediate in the NADH-driven hydride transfer process [136, 137]. Here, the conserved T66 entity in the reductase structure was shown to participate in modulation of the rate-limiting interconversion of the semiquinone forms [137]. Furthermore, mutation experiments verified the importance of the specific arrangement of R63, Y65 and S99 in the β-sheet barrel core of the flavoprotein in maintenance of FAD docking by electrostatic and H-bonding attraction of the si-face of the isoalloxazine ring [138, 139]. Similarly, the backbone amide nitrogen of M126 forms a hydrogen bond to the phosphate oxygen of the cofactor [140]. In addition, a series of residues including K110, S127, G179 and P275 were predicted to participate in the anchoring and proper positioning of the NADH electron donor [140, 141, 142, 143, 144, 145]. In this regard, the active-site C273 was considered to be critical for accurate orientation of the nicotinamide nucleus prior to hydride transfer [146]. Most interestingly, G179 and D239 were recognized to be required for efficient NADH/NADPH selectivity [144, 147].

Rapid electron transfer from NADH-cytochrome b5 oxidoreductase to b5 was shown to require N-terminal myristoylation of the flavoprotein to stabilize its orientation in the endoplasmic reticular membrane as a prerequisite for optimal productive encounter of the redox partners [148]. Here, circumstantial analysis implicated three reductase lysine residues hosted at positions 41, 125 and 163 in complementary charge pairing with the single exposed porphyrine propionate and a cluster of glutamate carboxyl groups at locations 43, 48, 49 and 53 (rat hemoprotein numbering) in the b5 polypeptide, surrounding the heme edge at a distance of ~12 Å [149, 150, 151, 152]. Qualitatively, the same b5 carboxyls were recognized to be essential for electrostatic interaction with prospective cationic groups in the alternate POR electron donor [153]. Employing the covalently cross-linked diflavoprotein/b5 heterodimer as a model for a functional electron-transfer complex, FMN depletion unveiled the cofactor-binding POR domain to be the active center for responsiveness to the hemoprotein. Here, lysines at positions 72, 74 and 75 are likely candidates for charge pairing with the b5 carboxyls [154]. As evidenced by the detrimental effect of removal of the N-terminus of POR on the functional coupling with b5, the intact hydrophobic tails of both redox proteins are required for efficient cross-talk between the partners facilitated by free lateral movement in the plane of the membrane [155]. Here, the nature of the system utilized for reconstitution of the matrix may steer the kinetics of intra- and intermolecular electron transfer [156]. Noteworthy, flash-induced b5 reduction by POR was found to proceed at a first-order rate about 10 % that measured with NADH-cytochrome b5 oxidoreductase [157, 158].

10.2.2.3 Characteristics of the Catalytic Cytochrome b5/Cytochrome P450 Redox Adduct

Cytochrome b5 plays a supportive role as a modifier of NADPH/POR-driven monooxygenations depending on the type of substrate and P450 species involved. For example, the presence of b5 invariably improves efficiency of product formation from methoxyflurane by CYP2B4 [159], fosters mephenytoin and chlorzoxazone turnover by CYP2C19 and CYP2E1 [160], and stimulates testosterone biotransformation by CYP3A4 [161].

The mechanism by which b5 impacts P450 activity has been extensively studied. When bound to ferric P450, the intermediate carrier elicits a low-to-high spin transition in the iron coordination sphere of the heme chromophore of the terminal pigment [162, 163]. Owing to the unfavorable discrepancy in the midpoint potential between the Fe3+/Fe2+ couples of b5 (−2.6 to +5.1 mV) and substrate-free ferric P450 (~ −400 to −300 mV), acceptance by the latter species of the first electron from the presumed donor protein can be excluded [33, 125]. In contrast, E0 for the labile oxyferrous P450 form is raised to 50 mV [164], permitting introduction of the second electron by ferrous b5 [111, 165] at a rate faster than autoxidation of the Fe3+-O2 intermediate associated with H2O2 release. This is expected to enhance economy of product formation at the expense of superoxide [166, 167]. It has to be noted that b5 may also exert non-redox, conformational effects on P450s. Thus, the modifier was shown to increase the steady-state level of the substrate-bound iron-oxo complex through lowering the energy of activation [162, 168] and to influence the rate of regeneration of ferric P450 from the oxygenated precursor as an index of the velocity of oxidative substrate turnover [169]. Precedent to this kinetic behavior is given by the action of apocytochrome b5 on the rate of productive decay of substrate-bound oxyferrous CYP101A1 [170]. Moreover, interaction of holo-/apo-b5 with CYP17 triggers rearrangement of the iron-dioxygen ligand necessary to awaken lyase activity [171, 172] or promotes repositioning of substrate to favor 16α-hydroxylation [173]. Generally, incorporation of heme-depleted b5 into reconstituted systems containing members of the CYP2 and CYP3 families was found to enhance typical catalytic activities to differing extents [174, 175]. Specific studies with the CYP4A7 species suggested apo-b5 to possibly alter the conformation of the substrate-binding pocket and/or accelerate product release [176]. In summation, these findings point at a dual role of b5 as an electron donor on the one hand and an allosteric effector on the other [177, 178].

The hydrophobic α-helical, membrane-spanning domain of b5 was demonstrated to play a dominant role in productive association with CYP2B4 [179]. However, introduction of alanines into the membrane anchor, expected to cause all amino acids distal to the insertion to undergo a 100° rotation, failed to disrupt any specific helix-helix interactions. This was interpreted to mean that b5/P450 binding proceeds through a nonspecific mechanism [180]. In contrast, the S90-D104 fragment, linking the heme domain of b5 with the C-terminal hydrophobic sequence, was postulated to restrict orientation of the donor/acceptor heme regions, facilitating formation of a functional complex [181]. Stability of the latter was shown to be granted by electrostatic and H-bonding attraction of complementary P450 residues by the invariant b5 amino acids Y30, E44, E48, D60 and T65, cooperating with the exposed heme propionate group [182, 183, 184].

10.2.3 Ferredoxins

Ferredoxins are small, soluble iron-sulfur proteins mediating electron transfer to P450s and other proteins such as nitrate and sulfite reductase. Classification of the intermediate carriers comprises different prototypes depending on the total number as well as Fe/S-proportion of the prosthetic clusters defining the active-site structure of the various electron shuttles [185]. In this regard, [Fe2-S2]-bearing ferredoxins, occurring in plants, bacteria and vertebrates, are of special interest [186]; the latter category includes both pro- and eukaryotic representatives [187]. Here, most extensive studies focus on the mammalian mitochondrial adrenodoxin (Adx) and the microbial putidaredoxin (Pdx) [188, 189], donating electrons to class I P450s [18]. Electron bridging requires prior transfer of reducing equivalents to ferredoxins by FAD-carrying NAD(P)H-ferredoxin reductases generally belonging to distinct types of unrelated protein families [40]. With respect to this, NADPH-adrenodoxin reductase (AdR) and NADH-putidaredoxin reductase (PdR) were uniformly assigned glutathione reductase-type redox proteins [190, 191].

10.2.3.1 Recognition of Adrenodoxin by Redox Partners

Site-directed mutagenesis experiments revealed the core domain of Adx, housing a single [Fe2-S2] cluster, to be mandatory for Adx/AdR association, while a second, acidic interaction site encompassing residues at positions 56–90 serves in docking of both AdR and P450s [192]. In accord with this, D72, D76 and D79 of Adx build up a tight H-bonding network with R211, R240 and R244 of AdR [193], but equally well operate in fixation of the steroidogenic CYP11A1 [194]. Noteworthy, the salt bridge between the invariant E74/R89 residues turned out to exert a principal stabilizing force impacting the orientation and redox properties of the iron-sulfur motif in parallel to AdR and CYP11A1 binding [195]. Genetic engineering of Y82 suggested the amino acid to be of importance in complex formation of Adx with CYP11A1 and CYP11B1, but to leave electron transfer unaffected [196]. In contrast, histidine at position 56 was recognized to control the integrity and ligand field of the protein region surrounding the [Fe2-S2] cluster [197]. Thus, H56T exchange was found to shift the redox potential of the wild-type Adx (−274 mV) to a value of −340 mV, causing a ~2.3-fold increase in the rate of CYP11A1 reduction [198]. Similarly, the vicinal T54 was recognized to modulate the protein’s redox state: conservative T→S replacement lowered E0 by ~60 mV as compared to the native ferredoxin without affecting AdR coupling and CYP11A1 reduction, though there was a marginal decrease in Kd for spectral binding of the hemoprotein [199, 200]. Of note, C-terminal truncation (Δ113–128) of Adx followed by S112W substitution was found to cause an 11-fold increase in the rate of CYP11A1 reduction associated with a 60-fold rise in the enzyme’s catalytic efficiency [200]. Finally, sequential deletion of residues E47, G48, T49, L50 and A51, located in a surface loop covering the iron-sulfur center, disclosed the domain to be crucial to regulation of the redox potential and functional coupling of AdR and CYP11A1 [201, 202].

As can be readily seen, the extensive spacial overlap of the interaction sites of Adx for AdR and P450 makes formation of a ternary complex improbable [203]. This view is substantiated by results from carbodiimide-mediated covalent crosslinking of Adx carboxylates to lysines on either AdR or CYP11A1. Structure-based assessment of the individual crosslink positions excluded a cluster model, but unequivocally suggested the ferredoxin to act as a mobile electron shuttle [204, 205]. Here, transport of reducing equivalents was hypothesized to proceed via both monomeric or dimeric Adx species [206]. The architecture of Adx-Adx assembly resulting in an asymmetric dimer was disclosed by crystal-based molecular modeling [207].

10.2.3.2 Molecular Recognition of Putidaredoxin by Redox Partners

Major driving forces in Pdx/PdR recognition were proven to encompass steric complementarity along with hydrophobicity and polarity. Thus, modeling studies combined with crystal-based mutagenesis experiments to modify both bulkiness of prospective key amino acids and their efficiency in charge pairing or van der Waals contacts identified the voluminous Y33 and R66 of Pdx, flanking the 365 Å2 protein-protein interface, to bind to R65/T66 and E335, respectively, in PdR. Substitution of the two ferredoxin residues with amino acids of lower molecular mass significantly increased the binding affinity of mutated Pdx to PdR, but drastically diminished kcat for electron transfer to the iron-sulfur cluster in view of moderate effects on E0 [208, 209]. This was interpreted to mean that the bulky side chains of tyrosine and arginine prevent tight docking of Pdx, so that transfer of reducing equivalents may occur via alternate pathways. In fact, optimal orientation for swift electron flow from FAD to the [Fe2-S2] center was predicted to be provided by interaction of W310 of PdR with D38 of the intermediate carrier [208, 209]. Moreover, ion pairing of the two residues is expected to lower the activation free energy for reduction of the metal cluster [189]. Evaluation of mutation and crosslinking data suggested the α-helical E72 of Pdx to form a salt bridge with K409 of PdR serving to establish and stabilize the electron transfer complex [209, 210], while the adjacent C73 seems to not only modulate the ferredoxin’s redox potential but to also define spacial approach of the subunits of the redox partners [208, 211]. Owing to flexibility of its aromatic ring, the C-terminal W106 of Pdx, oriented toward the center of the groove close to W330 of PdR [208], is thought to play a mediating and/or regulating role in the electron transfer process [212].

Importantly, the tryptophan at position 106 is of dominant importance in functional coupling of Pdx with the camphor-hydroxylating bacterial CYP101A1. Here, W106 is of higher relevance to transfer of the second electron to the oxyferrous hemoprotein than to donation of the first reducing equivalent to the ferric enzyme. This was argued to arise from the fact that the bulky, rigid indole ring of the tryptophan residue is apt to penetrate deep enough to approach the heme-binding loop of CYP101A1 [213] and induce structural changes required for acceleration of dioxygen activation, thus assisting the role of Pdx as an allosteric effector [189, 214]. It thus appears that the essential tryptophan exists in a conformational microheterogeneity [215]. In addition, D38 of the ferredoxin component was recognized to represent another hot spot in the two-step reductive event [214]. Starting from 3D modeling and molecular dynamics simulations, a series of amino acids such as D34 of the intermediate carrier were hypothesized to be likely candidates for intermolecular salt bridge formation, affording fixation of the Pdx/CYP101A1 complex [216]. In fact, D34N mutation was shown to depress catalytic efficiency (Vmax/Km) of the P450 system to a level 44 % of that found with the wild-type Pdx species [217]. Moreover, S42C exchange in the polypeptide clearly impacted donor/acceptor interaction [211].

Comparative evaluation of the general docking mode of the redox partners disclosed partial overlap of the proposed binding areas for PdR and CYP101A1 on the surface of the Pdx molecule, suggesting that the reductase and the hemoprotein cannot simultaneously interact with the ferredoxin [211]. This view seems to be in contrast to the competent function of a ternary PdR-Pdx-CYP101A1 fusion protein reported by others. Mobility of the fixed Pdx subunit of the latter construct appeared to be, nonetheless, high enough to pass electrons to exogenous native CYP101A1 introduced into the assay mixture [218]. In accord with this, analysis by optical biosensor techniques demonstrated the covalently immobilized three-component complex to exhibit only loose arrangement between Pdx and the terminal acceptor [219]. Also, studies on the kinetic behavior in dependence on the molar proportion of the individual redox partners supported the notion that Pdx acts as an electron transfer shuttle between PdR and CYP101A1 in analogy to the Adx-promoted route [220]. This raises the question as to what extent the bacterial CYP101A1-dependent system might be comparable to the mitochondrial CYP11A1-steered redox chain. Thus, inspection of the superimposed Adx/Pdx 3D structures, no doubt, permits one to discern significant homology (Fig. 10.5). Despite this, the two ferredoxins cannot substitute for each other in the two catalytic pathways owing to pronounced discrepancies in a series of functional determinants: (1) none of the acidic residues of Pdx corresponding to those vital to Adx fixation to CYP11A1 participate in intermolecular interactions with CYP101A1; (2) while T49 of Adx controls the redox dynamics of the iron-sulfur cluster, the equivalent S44 in Pdx fails to play such a role; (3) whereas the C-terminal aromatic tryptophan of Pdx is pivotal to tight CYP101A1 docking, the extended analogous region of Adx is deficient in such a P450-binding element. The interplay of these shortcomings causes Pdx and Adx to be unable to donate the second electron to the oxyferrous forms of the heterologous hemoproteins [221].
Fig. 10.5

Superposition of the three-dimensional structures of adrenodoxin (top) and putidaredoxin (bottom). The Adx (PDB ID: 1AYF) and Pdx (PDB ID: 1PUT) proteins, representing typical examples for vertebrate-type ferredoxins characterized by a sequence homology of ~35 %, display a similar planar geometry of the iron-sulfur cluster-containing region (spheres in yellow and blue). Generally, the overall folding topology of the α-helical and β-sheet elements shows a high degree of identity, with a 1.64 Å r.m.s. deviation between the two electron carriers (Reproduced from Ref. [221])

10.2.4 Unorthodox Electron Transfer Chains

Though P450s usually receive reducing equivalents from their dedicated redox partners, nonconventional electron transfer chains are frequently constructed to facilitate in vitro reconstitution of the donor/acceptor modules. For this purpose, the vertebrate-type ferredoxin/ferredoxin reductase components belong to the most frequently used surrogates of native intermediate carriers. Thus, the mitochondrial AdR/Adx couple turned out to interact with intact microsomal CYP1A1 such as to support erythromycin N-demethylation at higher efficiency compared to the inherent electron donor [222]. Similarly, Adx was demonstrated to cross-react with CYP2B enzymes, N-terminal hemoprotein truncation eliciting balanced reductive potency between the ferredoxin-promoted and P450 reductase-driven systems [223, 224]. Also, the truncated microsomal CYP17A1 and CYP21A2 proteins show higher steroid 17α-hydroxylase and 21-hydroxylase activity, respectively, with AdR/Adx compared to POR as the electron supplier [225]. Interestingly, CYP46A1, predominantly functional in cholesterol 24-hydroxylation in the brain, was found to interact with Adx as a redox component [226]. The mitochondrial carrier also sustains electron donation to the bacterial steroid 15β-hydroxylase CYP106A2 from Bacillus megaterium [227]. The system even operates at elevated catalytic capacity when AdR is replaced with NADPH-flavodoxin reductase from Escherichia coli to establish a novel robust redox chain [228]. Noteworthy, a mitochondrial ferredoxin reductase/ferredoxin unit from the fission yeast Schizosaccharomyces pombe was found to have >50 % sequence similarity with the mammalian AdR/Adx counterpart. The redox pair supports CYP11A1-catalyzed biotransformation of 7-dehydrocholesterol [229].

In addition, [Fe2-S2] proteins from non-mitochondrial sources have been demonstrated to transfer electrons to heterologous P450s. For example, plant-type ferredoxin and NADPH-ferredoxin reductase from spinach chloroplasts promote oxidative substrate turnover by microsomal CYP1A2 and CYP3A4 [230, 231] as well as 25-hydroxylation of vitamin D2 by CYP105A1 from Streptomyces griseolus [232]. Furthermore, bacterial electron transport systems such as the PdR/Pdx pair proved to foster β-carotene hydroxylation by the thermostable CYP175A1 species [233]. When working in concert with PdR, the microbial palustrisredoxin A factor readily feeds reducing equivalents to CYP199A2, preferentially metabolizing four-substituted benzoates [234]. Also, reconstitution of linredoxin and linredoxin reductase from a soil pseudomonad with CYP2B4 yields a collective efficiently metabolizing benzphetamine [235].

In some instances, Escherichia coli flavodoxin/flavodoxin reductase was detected to provide a basis for facile electron donation to microsomal P450s such as CYP1A2 [230], CYP3A4 [231] and CYP17A1 [236], but to equally-well pass electrons via a ping-pong mechanism to the microbial fatty acid oxidases CYP102A1 and CYP152A2 [237, 238]. Of note, a catalytically active system could also be established by employing flavodoxin reductase together with the unusual flavodoxin cindoxin from Citrobacter braakii as redox partners for CYP107H1 from Bacillus subtilis, having a role in biotin biosynthesis [19]. Finally, electron supply by POR from the yeast Candida apicola to the myristate-metabolizing CYP109B1 seems to be a unique case, where a eukaryotic diflavin reductase acts as a versatile electron donor to a bacterial hemoprotein [239]. In summation, cross-reactivity of electron carriers with a diversity of heterologous P450s can be reconciled with evolutionary conservation of a common functional domain architecture steering donor/acceptor interactions [32].

10.3 Topology of Critical Regions in P450s Dictating Interaction with Natural Redox Partners

10.3.1 Docking of NADPH-Cytochrome P450 Oxidoreductase

Data from chemical/immunochemical modification, molecular modeling and targeted mutagenesis were collated to generate an overall picture of key determinants in P450s responsible for POR fixation. Here, the N-terminal membrane-spanning signal anchor sequence of microsomal P450s seems to have a general role in protein-protein association: deletion of the membrane-immersed portion of CYP1A2 drastically decreases affinity for POR [230]. Genetic tailoring of a (Δ2–27)-variant of CYP2B4 proved to be detrimental to POR binding, resulting in a pronounced drop in the efficiency of electron transfer to the recipient [179, 240]. Chemical modification of the enzyme’s N-terminal α-amino group through covalent attachment of fluorescein isothiocyanate was recognized to compromise reductase docking via motional perturbation of the fluorophore-labeled region, eliciting a long-range effect on some distant patch involved in productive POR complexation [241]. Similarly, truncation of CYP2D6 was found to increase the Kd value for reductase binding by a factor of 11 [242]. Surprisingly, analogous manipulation of CYP2C3 and CYP2E1 failed to impede fixation of the flavoprotein [243, 244]. In contrast, the N-terminus of CYP6B33 from the insect Papilio multicaudatus is likely to maintain a protein fold obviously instrumental to communication with POR [245]. Also, construction of the (Δ1–66)-derivative of CYP52A3 from the yeast Candida maltosa was found to diminish reactivity toward POR [246].

To assess critical residues in P450s involved in the functional coupling of POR, CYP1A1 was covalently modified through treatment with acetic anhydride or an azido analog of benzphetamine. Selective blockage of four lysines at positions 97, 271, 279 and 407 was found to eliminate POR-dependent enzyme activity [247, 248]. This finding corresponds to results from studies with antibody targeted against a K271/K279-containing fragment of the hemoprotein, disclosing inhibition of metabolic turnover as the potential consequence of a rise in Km for POR [249]. In addition, attachment of 4,4′-dithiodipyridine to C293 in the CYP1A1 polypeptide was shown to be reversible upon incorporation of POR into the assay media, suggesting the residue to be located close to the reductase-binding motif [250]. This view receives support from antibody-directed suppression of substrate turnover following blockage of a region in the CYP1A2 homolog aligning with positions C293 to N301 in CYP1A1 [251]. Moreover, nitration of Y243 and Y271 in the CYP1A2 molecule was found to slow down electron transfer from POR to the acceptor [252]. Finally, site-directed mutagenesis helped verify prospective key players: Replacement in CYP1A1 of lysine at positions 271 and 279 with isoleucine caused a severe loss of responsiveness to POR for the hemoprotein [253]. Similarly, there was a 2- to 4-fold increase in the Kd value for POR anchoring when the basic lysines occurring at positions 94, 99, 440 and 453 in CYP1A2 were exchanged for an acidic residue [254, 255].

Within the plethora of drug-metabolizing P450s, inhibition of CYP2B1-mediated substrate oxidation by immunoprecipitation of the enzyme’s K122 to T231 sequence was shown to be less pronounced when antipeptide was added after reconstitution of the system with POR, proposing the epitope to be most likely engaged in POR association [256]. This concept is in line with R125 obviously having a critical role in this event [257]. Further lysine residues in CYP2B1, putatively serving as candidates for reductase recognition, reside at positions 251, 384, 422 and 433 [258]. Kinetic analysis of the chemically modified CYP2B4 analog in the absence and presence of protective amounts of POR disclosed lysines 139, 144, 251 and 384 to be in presumptive contact with the electron donor at a distance of about 3–4 Å [259]. Moreover, substitution of predominantly basic amino acids, hosted in the polypeptide fragment spanning residues R122 to K139, with the hydrophobic alanine entity drastically increased the Kd value for reductase binding to CYP2B4 [260]. Additional positively charged elements in the surface structure of the hemoprotein, identified by genetic engineering to promote electron flow from POR, include K225, H226, R232, R253 and H285 along with R422, K433 and R443 located in the vicinity of the heme edge [260, 261, 262]. On the other hand, a series of aromatic and hydrophobic amino acids such as F223, F227, F244, V267 and L270 were uncovered to participate in π-π-stacking and H-bonding interactions with POR [261, 263]. Of note, charge-reversal mutation K139E in the polymorphic CYP2B6.8 variant was found to impair functional complexation with POR [264]. This finding agrees with data from cross-linking experiments with the wild-type enzyme, disclosing competition of the latter with the synthetic D134-R140 peptide in reductase capture [265]. It should be mentioned that arginines at positions 139, 144 and 442, hypothesized to be beneficial to contacts with the electron donor in allelic CYP2C8 and CYP2C9 proteins as well as in CYP2C19, coincide with corresponding patches on CYP2B members [266, 267, 268]. Also, homology modeling of CYP2E1 in parallel with chemical inactivation by nitration of a series of tyrosines elucidated a close relationship between the FMN domain of POR and Y422 [269]. Noteworthy, C98W mutation in CYP3A4 was found to significantly hamper affinity to POR, associated with a 41 % diminution in the maximum rate of electron flow between the P450 and flavoprotein [270]. In addition, molecular modeling revealed the neighboring Y99 to be in close proximity to the cofactor-binding region of POR, while Y430 forms a hydrogen bond with an acidic reductase residue at a distance of 2.3 Å [271].

Inspection of microsomal P450s involved in the biosynthesis of natural products helped rescue further information about the architecture of donor/acceptor complexes. Thus, chemical and genetic modification of CYP17A1, lying at the crossroad of androgen and corticoid formation, unveiled the positively charged amino acids K326, K327, R347 and K358 to constitute part of the POR-contacting area [272, 273]. Similarly, a set of missense mutations at K121, R339, R341 and R356 provided clues to better understanding of the mode of interaction of POR with the steroid 21-hydroxylase CYP21A2 [274, 275, 276]. Finally, construction of a molecular model of the lanosterol 14α-demethylase CYP51F1 from yeast allowed identification of unique residues such as H101, K358, R426 and K433, serving as prospective sites for reductase association [277]. A compilation of the topological data for POR docking to the diverse P450s is given in Table 10.1.
Table 10.1

Prospective key amino acids of P450 enzymes governing interactions with redox partners: representative results from molecular modeling, genomic analyses and site-directed mutagenesis

CYP enzyme

Residue modified

Alignment positiona

Location in 2° structureb

Interacting redox partner

Refs.

POR

b5

Fdxc

51F1

H101

57

αB

+

  

[277]

1A1

K97

59

αB

+

  

[247]

1A2

K94

59

αB

+

  

[254]

17A1

K83

59

αB

 

+

 

[171]

101A1

R72

59

αB

 

+

+

[298]

11A1

K73

63

αB

  

+

[286]

101D1

R77

63

αB

  

+

[304]

1A2

K99

64

αB

+

  

[254, 255]

17A1

K88

64

αB

 

+

 

[171]

2B1

K122

97

αC

 

+

 

[280]

2B4

R122

97

αC

+

+

 

[260]

11A1

K109

97

αC

  

+

[286]

101A1

R109

97

αC

  

+

[299]

119

R109

97

αC

  

+

[303]

3A4

K127

98

αC

 

+

 

[283]

11A1

K110

98

αC

  

+

[286]

2B1

R125

100

αC

+

+

 

[257]

101A1

R112

100

αC

  

+

[299, 300, 301]

101D1

R113

100

αC

  

+

[304]

2A5

R129

101

αC

 

+

 

[278, 279]

2B4

R126

101

αC

+

+

 

[260]

21A2

K121

101

αC

+

  

[274]

2B1

S128

103

αC

 

+

 

[281]

2B2

S128

103

αC

 

+

 

[281]

2B4

S128

103

αC

 

+

 

[281]

102A1

L104

104

αC

+

  

[354]

2B4

R133

108

αC1

+

+

 

[260]

2B4

F135

110

αC1

+

+

 

[260]

2B4

M137

112

αC1

+

+

 

[260]

2B4

K139

113

αC1

+

+

 

[259, 260]

2B6

K139

113

αC1

+

  

[264]

11A1

K126

114

αC1

  

+

[286]

2C9

R144

118

αD

+

  

[267]

11A1

K145

133

αD

  

+

[286]

2B4

H226

197

αG

+

+

 

[260, 261]

2B4

F227

198

αG

+

  

[261]

2B4

R232

203

αG

+

  

[261]

101A1

K197

209

αG

  

+

[302]

2B4

F244

215

αG

+

  

[261]

1A1

K271

221

αG

+

  

[247]

2B1

K251

222

αG

+

  

[258]

2B4

K251

222

αG

+

  

[259]

1A2

Y271

224

αG

+

  

[252]

2B4

R253

224

αG

+

  

[261]

1A1

K279

229

αG-αH

+

  

[253]

2B4

V267

236

αH

+

  

[263]

2B4

L270

239

αH

+

  

[263]

1A1

C292

241

αH-αI

+

  

[250]

11A1

K267

245

αH-αI

  

+

[286]

11A1

K270

248

αH-αI

  

+

[286]

2B4

H285

250

αI

+

  

[262]

17A1

K326

288

αJ

+

  

[272]

17A1

R347

308

αJ‘

+

+

 

[273, 284]

21A2

R339

308

αJ‘

+

  

[276]

21A2

R341

310

αJ‘

+

  

[276]

51F1

K358

310

αJ‘

+

  

[277]

11A1

K338

315

αK

  

+

[286, 287]

27A1

K354

315

αK

  

+

[296]

199A2

R285

315

αK

  

+

[305]

11A1

K342

319

αK

  

+

[286]

17A1

R358

319

αK

+

+

 

[273, 284]

27A1

K358

319

αK

  

+

[296]

21A2

R356

325

αK

+

  

[276]

1A1

K407

349

β2(2)-β1(3)

+

  

[247]

2B1

K384

349

β2(2)-β1(3)

+

  

[258]

2B4

K384

349

β2(2)-β1(3)

+

  

[259]

51F1

R426

377

MR

+

  

[277]

3A4

K421

380

MR

 

+

 

[283]

27A1

R418

380

MR

  

+

[296]

11A1

K405

383

MR

  

+

[292]

101A1

K344

383

MR

 

+

+

[298]

51F1

K433

384

MR

+

  

[277]

2B1

K422

386

HBR

+

  

[258]

2B4

R422

386

HBR

+

  

[260]

2E1

Y422

386

HBR

+

  

[269]

102A1

Q387

387

HBR

+

  

[354]

1A2

K440

388

HBR

+

  

[254]

3A4

Y430

388

HBR

+

  

[271]

2E1

K428

391

HBR

 

+

 

[282]

1A2

K453

397

HBR

+

  

[255]

2B1

K433

397

HBR

+

  

[258]

2B4

K433

397

HBR

+

+

 

[260]

2E1

K434

397

HBR

 

+

 

[282]

1A2

R455

399

HBR

+

  

[254]

11A1

R426

404

αL

  

+

[292]

24A1

R466

404

αL

  

+

[294]

2B4

R443

407

αL

+

  

[260]

2C19

R442

407

αL

+

  

[268]

17A1

R449

407

αL

 

+

 

[284]

101D1

R371

407

αL

  

+

[304]

199A2

L369

408

αL

  

+

[305]

aPositions were determined by screening the sequences of the target P450 enzymes against the crystal structure of substrate-bound CYP102A1 (PDB ID: 1ZO9) as described previously [277, 307]

bAllocation of the alignment positions to definite domains of α-helical or β-sheet structure is based on the CYP102A1 architecture [314]. MR meander region, HBR heme-binding region

cThe category of ferredoxins includes Adx, Arx, Pdx and Pux

10.3.2 Docking of Cytochrome b5

In cases where P450s exhibit an obligatory requirement for electron donation by b5 to maintain optimal rates of substrate turnover, structural integrity of the hydrophobic tail portion of the oxidases seems to be pivotal to productive donor/acceptor coupling. For example, optical biosensor studies with CYP2B4 lacking amino acids 2–27 disclosed removal of the signal anchor to result in defective binding of the intermediate carrier accompanied by a pronounced drop in the reductive force [179, 240]. Apart from this, circumstantial exploration of a set of CYP2 members helped ascertain an array of critical b5-docking entities sitting remote from the enzymes’ N-terminus. Thus, strongly perturbed donor anchoring upon generation of the R129S derivative of CYP2A5 suggested the RRFS fragment in the polypeptide chain to be a key recognition motif [278]. This conclusion nicely coincides with the fact that the homologous CYP2A4, bearing a R129S point mutation, fails to stimulate substrate oxidation [279]. Interestingly, site-specific attack on K122, R125 and S128 in the CYP2B1 polypeptide by immunochemical manipulation or protein kinase-mediated phosphorylation was found to be competitively antagonized by the presence of b5, suggesting these residues to be in contact with the electron donor [280, 281]. Moreover, elements R122, R126, R133, F135, M137, K139, H226 and K433, selected by computer docking of a CYP2B4 model, were substituted with alanine to evaluate the function of the amino acid side chain distal to the β-carbon. All the mutants tested displayed diminished ability to bind b5 [260]. Genetic engineering was also employed to confirm the biological importance of K428 and K434 in CYP2E1/b5 complexation [282].

Studies extended to other P450 families verified sites K127 and K421 on CYP3A4 to be essential for efficient b5 coupling [283]. Moreover, impairment of the fundamental chemistry by introduction of mutations at positions 83, 88, 347, 358 and 449 in CYP17A1 was demonstrated to hamper propensity for 17,20-lyase activity by disrupting responsiveness to the b5 component [171, 273, 284]. Table 10.1 provides a synopsis of key amino acids in P450s governing interaction with b5.

10.3.3 Docking of Ferredoxins

Use of a specific fluorescence probe localized the heme group of the mitochondrial CYP11A1 protein ~26 Å remote from the binding surface for adrenodoxin (Adx) [285]. Here, basic residues K73, K109, K110, K126, K145, K267, K270, K338 and K342 on the mature hemoprotein form were substantiated to govern reactivity toward Adx by the ferredoxin’s ability to act as an almost complete protector against the lysine-modifying agents, succinic anhydride or fluorescein isothiocyanate, employed for enzyme engineering [286, 287]. Two additional lysines corresponding to K377 and K381 in the precursor form of steroidogenic CYP11A1 were identified by site-directed mutagenesis as also being vital to Adx association. Estimated Kd values for donor docking increased about 150- to 600-fold compared to the wild-type enzyme depending on the particular lysine substitute [288]. This finding fits data from specific chemical labeling of lysines in the peptide comprising amino acids M369 to K381 in the CYP11A1 molecule, eliciting a drastic fall in responsiveness to the electron-supplying factor [289]. Of note, point mutation R→C at position 366 in CYP11B1, corresponding to K377 in the CYP11A1 congener, was detected to give rise to breakdown of the catalytic efficiency of 11β-hydroxylation to a level ~25 % that of the native protein. This has been interpreted to mean that a change to cysteine eliminates a positive charge and leaves a cove on the enzyme’s surface, most likely impacting Adx fixation [290]. Interest also focused on residue C264 lying proximate to K267 in the so-called “hinge” region. Indeed, chemical blockage of the surface cysteine was found to hamper CYP11A1-promoted turnover through curtailing the catalyst’s capacity to interact with Adx [291]. Moreover, biochemical and molecular modeling studies based on the crystal structure of the redox partner jointly supported the concept that K405 and R426 (numbering of the mature hemoprotein form) participate in electrostatic contacts with Adx [292]. There seems to exist an interplay between the latter amino acid and the conserved vicinal E429 residue responsible for fine tuning of the stability of the assembled complex [293]. Crystallographic analysis of the 24-hydroxylase CYP24A1 from rat again revealed structural elements K378 and K382, aligning with lysines at positions 377 and 381 in bovine CYP11A1, to operate as key players in Adx recognition. In addition, the invariant R466, located 8–10 Å remote from the conserved lysines, was assigned a dominant function in ferredoxin-driven electron transfer [294]. The critical arginine aligns with R426 in CYP11A1 and R458 in the murine CYP27B1. In fact, R458Q substitution was detected to induce a 36-fold rise in the apparent Km value for Adx associated with a drastic decrease in electron pressure [295]. Finally, introduction of the K354A/K358A/R418S triad into CYP27A1 involved in bile acid biosynthesis was shown to be destructive to ferredoxin binding [296].

Epitope mapping, carried out with bacterial CYP101A1 from Pseudomonas putida in the presence of a set of antigenic peptides directed against areas presumed to be of functional relevance, suggested regions spanning residues 63–72 and 108–117, respectively, to potentially participate in putidaredoxin docking [297]. This view was underpinned by the severe loss of reactivity toward Pdx upon creation of hemoprotein variants bearing non-ionic amino acids in place of the positively charged arginine at positions 72, 109 and 112 [217, 298, 299]. It should be emphasized that ferredoxin binding to R112 has been recognized to also be beneficial to intracomplex electron transfer to the ferric heme iron-oxo species [300, 301]. Moreover, the ability of the intermediate carrier to shield K197 in the P450 molecule from attack by chemical modifiers qualifies the lysine residue as part of the Pdx recognition site [302]. Similarly, reversal of the cationic charge by K344E mutation was demonstrated to cause perturbation of donor docking [298]. Interestingly, CYP119A1 from thermophilic Sulfolobus acidocaldarius utilizes Pdx as the electron supplier. Here, D77R mutation of the hemoprotein was detected to markedly enhance fixation of the redox partner and stimulate electron flow by a factor of about 5 compared to the parent enzyme, obviously eliminating a potentially repulsive protein-protein interaction [303]. The repellent effect of D77 thus might serve in proper Pdx orientation.

Other bacterial P450s receive electrons via [Fe2-S2]-type ferredoxins genomically associated with the individual oxidases. For example, evaluation of the electrostatic surface profile of CYP101D1 from the oligotrophic Novosphingobium aromaticivorans suggested amino acids such as R77, R113 and R371 to contribute to specificity in [2Fe-2S]-type ferredoxin (Arx) recognition [304]. Furthermore, the benzoic acid-oxidizing CYP199A2 from Rhodopseudomonas palustris was recognized to carry two surface hot spots presumed to be significant factors in steering cross-reactivity of ferredoxins. Thus, the presence of R285 as well as charge reversal at L369 were hypothesized to be responsible for preferential functional coupling of the physiological redox partner palustrisredoxin compared to the heterologous Pdx [305, 306]. A summary of data for ferredoxin docking to vertebrate-type P450s is presented in Table 10.1.

10.3.4 Overall Architecture of Redox Domains and Mechanism of Electron Donor Docking

Increasing interest in elucidation of the molecular mechanism of electron transfer in P450 systems creates fundamental demand for visualization of the architecture of donor-binding sites ruling catalytic potency of the enzymes. To accomplish this goal, homology modeling has to be carried out using sophisticated strategies for construct building. Judging from data for root mean square (r.m.s.) deviation of critical Cα atoms and φ/ψ-angle distribution, comparative alignment by knowledge-based techniques of P450s from different phyla with the bacterial CYP102A1, having its 3D structure determined, suggested the microbial enzyme to be a robust template for elucidation of structure-function relationships [277, 307]. Thus, mapping of key amino acid residues from microsomal, mitochondrial and bacterial hemoprotein species recognized to contribute to redox partner interactions (Table 10.1) onto the CYP102A1 scaffold yielded a scenario (Fig. 10.6) describing the general spatial distribution of contact sites [32].
Fig. 10.6

Generalized molecular model featuring critical surface sites in P450s operating in recognition and binding of redox proteins. The composite profile was built by mapping the topological data of key determinants steering electron donor fixation onto the substrate-bound CYP102A1 template. The color code denotes: yellow spheres, POR-binding sites; blue spheres, b5-binding sites; green spheres, sites common to POR and b5; red spheres, Fdx-binding sites; purple spheres, sites common to Fdx and b5. For top and bottom views, the coordinates of the images were rotated by 90° in the x-axis (Data taken from Ref. [32])

As can be seen, the majority of points presumed to dictate contact with redox proteins cluster close to the center of the proximal face of the hemoprotein model. Highest density of binding sites, amounting to 47 % of the total number of key players, is found in the triad formed by α-helical structures C/C1, bordering the core fold on the top, the G-helical fragment, located more in the periphery of the P450 molecule, and the heme-binding region. The remaining interaction sites appear to be of minor importance, each housing but 5–8 % of the overall volume of anchoring elements (Table 10.1). Surprisingly, the population of functional determinants in the various target P450s, residing in the three preeminent donor-docking epitopes, displays a very low to moderate extent of conservation ranging from 9 % to 27 %. This might arise from the need for conformational flexibility to enable encounter with heterologous redox proteins. Indeed, ~38 % of the contact sites harbored in helices C/C1/G and the heme-binding domain have overlap of POR fixation with b5 recognition. This behavior agrees with the ability of increasing amounts of b5, integrated into assay media containing a constant level of POR, to gradually transform the biphasic kinetic tracings, prototypic of NADPH-driven P450 reduction, to a sluggish monophasic reaction as is characteristic of electron donation by b5 [308, 309]. This lends support to the notion of a functional antagonism between the two redox proteins. On the other hand, b5 fails to interfere with nonproductive physical anchoring of reductase to P450, as evidenced by visible difference spectrometry [308]. This seems to hint at functional diversification of the POR-docking loci [310] potentially acting in substrate-induced cooperativity [241]. Though overlap of regions involved in POR and ferredoxin association is lacking, the redox domain architecture (Fig. 10.6) displays epitopes fostering binding of the two carrier species to cluster in close proximity to each other in helices B/C and the H-I interhelical loop, possibly caused by certain analogy in the structural organization of the electron transfer proteins [185]. Similarly, evolutionary commonality induces joint contact points for b5 and ferredoxins on the proximal face of P450s constituted by portions of helices B, C, K and the meander stretch (Table 10.1). It should be mentioned that the crystal structure of an archetypal b5 homolog isolated from a bacterial strain has been identified [311]. This prompts one to speculate that b5-type proteins may act as natural electron donors also to certain microbial P450s.

Evaluation of the array of data summarized in Table 10.1 disclosed 81 % of the overall population of amino acids predicted to operate in redox partner binding in the various target P450s to belong to the category of positively charged entities, about two thirds of the reactants being represented by lysine residues and one third by arginines. Indeed, calculation of the electrostatic surface potential for a series of P450s showed the dipole moment of the hemoproteins to be oriented such as to help direct the intermediate carriers toward a patch of positively charged elements on the proximal face [312, 313, 314]. This behavior underpins salt-bridge formation with carboxylates in the donor proteins (see Sect. 10.2) to be the most salient driving force in complexation, as exemplified by the allocation of interfacial residues involved in the CYP3A4-b5 encounter [283] depicted in Fig. 10.7. In agreement with this principle, charge shielding by high concentrations of mobile ions was shown to elicit disintegration of donor/acceptor anchoring associated with a drop in electron flow [255, 293, 298]. Moreover, ~10 % of the key players bear a polar side group serving in generation of a flexible H-bonding link to some basic group(s) in the intermediate carriers, with tyrosines presumably being favored mediators of weakly polar inter-residue contacts [252, 269, 271].
Fig. 10.7

Functional importance of electrostatic interactions between the protein surfaces of cytochrome b5 and CYP3A4. The monooxygenase and the electron carrier are presented in white and green, with their heme groups being shown in red and orange, respectively. The interacting residues on the redox partners are depicted in magenta and blue, while the critical R446 is colored golden. As is evident, b5 approaches the B-B′ loop region and helix C of CYP3A4 via helices α4 and α5. Protein domains on the oxygenase far from the docking surface are truncated (Reproduced from Ref. [283])

Since electrostatic phenomena, no doubt, prevail in functional coupling of redox partners, it does not seem surprising that only a minority (~9 %) of the total of sites attracting electron donors can be assigned to the class of lipophilic amino acids largely accommodated in helices C1 and G. Here, aromatic and aliphatic representatives cooperate in π-π-stacking and van der Waals interactions with reactants in the diverse redox proteins [260, 261, 263, 305].

10.3.5 Factors Impacting Organization of Protein-Protein Association

10.3.5.1 The Role of Phospholipids

The phospholipid matrix serving in insertion and assembly of the components of the P450-dependent redox machinery was detected to strongly impact efficiency of electron transport in microsomal and mitochondrial systems by providing structural features permitting improved recognition, ordering and alignment of redox partners [315]. In this respect, the synthetic dilauroyl phosphatidylcholine as well as natural phospholipids were shown to decrease the apparent dissociation constant for P450/POR complexes to an extent depending on both the chain length of the lipids and the mode of reconstitution, yielding either micellar or vesicular systems [316, 317, 318]. Owing to predominance of positive charges on the proximal profile of P450s (see above), mixtures containing anionic lipids such as phosphatidylserine were found to favor P450/POR association by forcing the proteins into correct orientation toward each other [319, 320, 321]. Facilitated donor/acceptor binding appears to generally require prior phospholipid-induced relaxation of the tight multimeric P450 aggregates [322]. Indeed, displacement of the P450 oligomerization equilibrium toward monomers by use of a nanoscale construct bearing a palmitoyl-oleoyl phosphatidylcholine bilayer drastically improved flavoprotein-promoted P450 reducibility [323]. Collectively, phospholipids were recognized to act as allosteric effectors eliciting conformational alterations in P450s associated with an increase in α-helical content of the hemoproteins. This fosters functional coupling of different types of electron carriers [324, 325]. Thus, lipid was demonstrated to also modulate affinity of b5 for CYP2B4 [326]. Conversely, b5 binding to the enzyme caused a ~2-fold rise in reactivity of phosphatidylcholine to CYP2B4 [327]. Similarly, cholesterol lowers Kd for Adx docking to cardiolipin-saturated CYP11A1 by a factor of up to 20, while the ferredoxin, in its turn, improves cholesterol binding to steroidogenic CYP11A1 [328].

10.3.5.2 The Role of P450-P450 Aggregation

Formation of hetero-oligomers of P450 is well documented and may play a decisive role in regulatory mechanisms of electron transfer [329]. In fact, the combined presence of CYP1A2 and CYP2B4 reconstituted with POR in the same phosphatidylcholine vesicles provides conclusive evidence from changes in the enzyme-specific monooxygenase activities that the CYP1A2 moiety of the heteromeric P450 complex generates a high-affinity reductase adduct more effectively competing for the redox protein than CYP2B4 [330]. Comparable results were obtained when the CYP2E1/CYP2B4 pair was embedded into a phospholipid matrix in the presence of POR to probe competition for the reductant. Here, low levels of CYP2E1 turned out to cause a 23-fold increase in the apparent Km value of CYP2B4 for the donor protein, while the analogous Km of CYP2E1 for POR decreased significantly, allowing CYP2E1 to outact CYP2B4 [331]. Of note, CYP2A6/CYP2E1/POR co-expression in microsomal membranes disclosed the presence of a prototypic CYP2A6 substrate to impair electron flow to CYP2E1, suggestive of a regulatory function of substrate in P450 aggregation [332]. This view is substantiated by drug-drug interactions occurring as the output of competition for the ancillary POR enzyme of co-reconstituted P450 couples such as CYP2C9/CYP2C19 or CYP2D6/CYP3A4 [333, 334]. Furthermore, the formation in liposomal membranes of an equimolar complex between the mitochondrial CYP11A1 and CYP11B1 enzymes was found to have a stimulatory effect on the CYP11B1-dependent 11β-hydroxylase activity as the consequence of a conformational alteration, corresponding to changes in the Km value for Adx [335]. A mathematical model taking account of the possible existence of multiple types of P450-based dimer formations was developed to explore the most probable mechanism(s) of such interactions in more detail [336].

One would be remiss without mentioning that a fraction of P450s integrated into membranous systems may also exist as homo-oligomers. Here, formation of large aggregates causes P450 immobilization to an extent depending on the lipid-to-protein ratio [337, 338]. Interestingly, incorporation of POR or b5 was shown to readily disrupt the aggregation state of P450s, when in a membrane, via transient complexation with the monooxygenases. This might influence the amount of productive donor/acceptor adducts determining catalytic activity [339, 340]. Again, substrate may interfere with the docking events to modulate reactivity of the redox partners [33, 177].

10.4 P450/Redox Partner Fusion Enzymes

10.4.1 Natural Fusion Proteins

Among fusion enzymes, the cytosolic CYP102A1 from Bacillus megaterium represents a unique self-sufficient flavohemoprotein catalyzing (ω–n)-hydroxylation of medium- to long-chain saturated fatty acids [341]. Owing to its soluble nature and applicability as an excellent paradigm for the understanding of structure/function relationships in class II-type P450s, CYP102A1 represents the most extensively studied member of the CYP102A subfamily consisting of a large number of relatives, though only four additional homologs, namely CYP102A2/A3/A5 and A7 from diverse Bacillus strains, have so far been characterized. Here, comparison of the polypeptide structures revealed some deviations in active-site architecture [342, 343, 344].

The CYP102A1 enzyme is composed of an N-terminal heme domain connected via a short protein linker with a eukaryotic-like diflavin reductase module bearing one equivalent each of FAD and FMN [345]. Availability of the crystal structure of the FAD/NADPH-binding domain helped identify sites involved in NADPH fixation such as S965, R966, K972 and Y974 [346]. Noteworthy, the side chain of W1046 shields the FAD isoalloxazine ring from NADPH, and motion of this residue drives pyridine nucleotide specificity to the formation of an FADH2-NAD(P)+ charge-transfer intermediate [347]. Aromatic stacking with W854 and Y860 was shown to stabilize the FAD cofactor, while amino acids at positions 729–743 have the potential to make contacts with the cognate FMN domain [346]. Due to the dimeric nature of CYP102A1, the obligatory electron tunneling route traverses both constituents of the dimer during a single turnover by switching from the FAD-binding site of one monomer to the FMN domain of the other one prior to passing on to the terminal acceptor [348]. Interestingly, modulation of the electrostatic microenvironment of the FMN-docking pocket, housing critical residues Y536 and G570 [349], by unusual integration of positively charged lysines at positions 572 and 580 as well as decreased flexibility of the short cofactor-binding loop were presumed to be jointly responsible for the observed repression of the neutral, blue FMN semiquinone radical paralleled by stabilization of the red, anionic hydroquinone species. This was shown to be coupled with a change in the E0 values of the redox pairs securing electron flow to the heme unit [350, 351, 352]. In this regard, W574, located in the FMN domain, was demonstrated to provide a direct through-bond electron transfer pathway including P382 and C400 in the heme-binding peptide [349, 350], while the highly conserved W96 turned out to have a function in heme association and control of the spin state of the iron [353]. Moreover, the area around L104 and Q387 in the intact heme/FMN-binding fragment (Fig. 10.8) revealed to be vital to efficient functional association of the partners [354]. It has to be mentioned that a soluble form of microsomal b5 was found to also undergo tight binding to CYP102A1, eliciting a low-to-high spin transition in the enzyme’s heme iron. This suggested the electron donor to occupy a contact site on the proximal face of the P450 heme that overlaps with that for the FMN domain of the diflavin reductase [355].
Fig. 10.8

View of the 3D structure of the complex between the heme- and FMN-binding domains of bacterial CYP102A1. The flavin-binding domain (green) hosting the FMN cofactor (yellow) is physically linked on the same polypeptide to the region (blue) surrounding the iron-porphyrin macrocycle (red). In this complex, the dimethylbenzene ring of FMN is oriented perpendicular to the heme plane at a distance of ~18 Å (Data taken from Ref. [350])

Genetic exploration of the fungus Fusarium oxysporum revealed the existence of a loosely membrane-associated, self-sufficient flavocytochrome termed CYP505A1, sharing ~41 % sequence identity with the P450 moiety of the bacterial CYP102A1 counterpart [356]. The enzyme catalyzes pyridine nucleotide-driven (ω-1)- to (ω-3)-hydroxylation of saturated C9 to C16 fatty acids [357]. The reductase unit in the primary CYP505A1 structure, having 35 % sequence identity with that of CYP102A1, was shown to be fixed to the heme region via a linker consisting of 20 amino acids of mainly hydrophilic character [356]. Noteworthy, 28 % of the residues hosted in fractions forming the NADPH/FAD- and FMN-binding domains disclosed to be invariant, with hydrophilicity clearly prevailing in the bond-making events [356]. Here, electron transfer was shown to be strongly stimulated by the presence of substrate [358]. Another self-sufficient member of the CYP505 family, classified CYP505B1, was isolated from the ascomycete Fusarium verticillioides and found to participate in the biosynthesis of the polyketide mycotoxin fumonisin. The flavohemoprotein displays 41 % sequence identity to CYP505A1 and 33 % identity to CYP102A1, with the putative cofactor-docking regions being arranged in the same order as in the homologs cited [359].

In addition, new types of P450-redox partner fusions have been unveiled. An example is bacterial P450 XplA (CYP177A1) from the Rhodococcus rhodochrous strain 11Y, catalyzing reductive denitration of the military explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) [360]. The enzyme has an unusual structural organization comprising the N-terminal P450 heme domain fused to a flavodoxin unit [361, 362]. The latter was shown to contain most of the elements of a signature typical for FMN binding [360]. Unexpected features include the low affinity of the non-covalently bound FMN to its docking site (Kd = 1.09 μM) compared to reactivity of the cofactor toward flavodoxins from other bacterial sources and the strong positive shift of the redox potential of the FMN semiquinone/hydroquinone couple \( \left({E}_0^{\prime }=-172\kern0.24em \mathrm{mV}\right) \), being the likely electron donor to the XplA heme [363].

Efficient RDX degradation was shown to require expression of the partnering reductase XplB. The FAD-containing carrier transfers reducing equivalents to the XplA-FMN in a 1:1 complex with high specificity for NADPH as the electron source [362, 363]. Here, collision of the two subunits represents a rate-limiting step. Of note, the deduced amino acid sequence of XplB has 42 % similarity to bovine mitochondrial AdR [360]. In accord with this, the XplA flavodoxin domain is capable of receiving electrons also from ferredoxin reductase before transferring them to the P450 heme [364].

Circumstantial exploration of the bacterial genome sequence library uncovered a completely novel class of self-sufficient P450 systems, representing a distinctive community of enzymes C-terminally fused to a phthalate dioxygenase reductase (PDR) module as the redox partner. The latter is folded into three domains involved in NADH/FMN binding and docking of the [Fe2-S2] cluster [365]. Thus, CYP116B1 from Cupriavidus metallidurans, a thiocarbamate herbicide-oxygenating fusion protein, displays stoichiometric binding of both FMN and the iron-sulfur center, electron transfer being supported by NAD(P)H with clear dominance of the triphosphopyridine nucleotide [366]. Similarly, CYP116B2 from a Rhodococcus species was demonstrated to be composed of an N-terminal P450 moiety separated by a short segment of about 16 amino acids from the reductase-like fragment, sharing 34 % sequence identity with the PDR family [367]. Closer investigation of the electron-supplying subunit predicted P578 to contact pyridine nucleotides, with NADPH having a ~500-fold preference over NADH in terms of the estimated Kd values [367, 368]. Moreover, the stretch spanning residues S532 to S536 was found to conform to the consensus motif for binding of the phosphate group of FMN, while a cluster of four highly conserved cysteines at positions 722, 727, 730 and 760 constitutes a [Fe2-S2] ferredoxin-type center [367]. The reduction potentials of the FMN semiquinone/hydroquinone and FeS entities were calculated to be approximately −270 mV and −214 mV, respectively [369]. Substrate screening for CYP116B2 revealed the enzyme to mediate dealkylation of substituted aromatic alkyl ethers, catalytic efficiency being higher with compounds bearing a shorter alkyl chain [370]. In addition, a new self-sufficient member of the CYP116 family was identified in Rhodococcus ruber. The fusion protein was recognized to have >90 % amino acid sequence identity to CYP116B2 and to consist of a heme domain, an FMN-hosting region and an iron-sulfur unit. In the presence of NADPH, the enzyme shows hydroxylase activity toward polycyclic aromatic hydrocarbons such as naphthalene or fluorene [371].

10.4.2 Artificial Self-Sufficient Fusion Proteins

The catalytic diversity of P450s has high potential for biotechnological exploitation. However, industrial application is frustrated by the need of costly NAD(P)H cofactors, cumbersome reconstitution of auxiliary electron donor systems and fairly low metabolic turnover. To simplify the procedure and improve the catalytic outcome, the natural P450/redox partner fusion proteins addressed above may represent excellent paradigms for the sculpturing of man-made chimeric analogs of desired autonomic electron transport [372]. This requires cDNA shuffling to design assembly of redox chain building blocks on a “molecular Lego” principle [373]. Also, a versatile “drop-in” vector for rapid creation of self-sufficient P450s has been developed [374].

10.4.2.1 P450/Diflavin Reductase Fusion Enzymes

A useful tool for the generation of simplistic P450 redox systems is featured by covalent fixation of the hemoprotein portion to a POR-like unit with the aim to construct the most suitable fusion [375]. In this way, the (Δ1–41)-truncated reductase moiety from yeast was genetically attached to rat CYP1A1. The construct displayed rotational mobility of the P450 fragment higher than that of CYP1A1 alone [376]. In the presence of NADPH, the rate of reduction of the substrate-bound fusion enzyme was found to be >50 s−1, suggesting that electrons were rapidly transferred from the cofactor through FAD and FMN to the heme iron [377]. Similarly, human CYP1A1 was connected to N-terminally truncated rat POR via a Ser-Thr dipeptide linker. Activity toward resorufins was shown to be 11- to 22-fold higher compared to the control [378]. Of note, human CYP1A2 genetically engineered with yeast reductase proved to be about twice as efficient in oxidative ethoxyresorufin biotransformation relative to the CYP1A1 fusion system [379]. Moreover, a fused construct derived from the cDNA for canine CYP2B11 in tandem with the code for the modified rat oxidoreductase exhibited an androstenedione metabolite profile very similar to that found with the reconstituted components [380]. The same approach was adapted to arrange chimeras produced by joining the C-terminus of mammalian CYP2C11 or CYP2D6 to the cytoplasmic domain of the cognate flavoproteins via a dipeptide linker. In either case, molecular organization seemed to be suboptimal, as judged from comparison of the kcat values for substrate turnover with those of the non-fused systems [381, 382]. Similar observations were made upon linkage of human CYP3A4 to rat POR. Addition of excess exogenous POR and b5 to the reaction mixtures were found to drastically enhance the rate of testosterone 6β-hydroxylation [161]. Deficiency in catalytic capacity of the fused construct may arise from the fairly short linker region restricting flexibility in orientation toward each other of the functional interfaces of the CYP3A4 and POR modules required to permit swift electron transfer. Optimization was achieved by engineering a number of triple adducts, among which the CYP3A4/reductase/b5 product turned out to reflect the most appropriate ordering for high activity compared to the reconstitution premixes [383]. Though fusion of rat CYP4A1 with the native reductase unit gave a biocatalyst mediating lauric acid ω-hydroxylation at a rate threefold higher than that determined in reconstitution assays, metabolic capacity was not fully exhausted: supplementation with purified flavoprotein/b5 strongly stimulated fatty acid consumption by potentially increasing collision frequency of the redox partners [384, 385].

The fusion strategy was also extended to microsomal steroidogenic P450s. Thus, the multifunctional CYP17A1 domain of different mammalian species was connected to a truncated form of yeast or rat reductase to yield a self-contained unit characterized by 17α-hydroxylase and 17,20-lyase activity, promoting biotransformation of progesterone and pregnenolone to the corresponding C19-derivatives [386, 387]. Here, the length and amino acid sequence of the hinge region between the redox components was demonstrated to play a decisive role in efficient intramolecular electron transfer [388]. Of interest, donation to the fused adduct of reducing equivalents by exogenous b5 was shown to boost the lyase pathway to an extent depending on the genetic ancestry of the CYP17A1 moiety examined [389]. Similarly, linkage of bovine CYP21A2 to N-terminally modified yeast POR gave a flavohemoprotein driving conversion of 17α-hydroxyprogesterone to 11-deoxycortisol at a catalytic efficiency two- to four-times greater than that found with the reconstituted CYP21A2 redox chain [390]. Surprisingly, a mitochondrial form of rat CYP27A1, when fixed to a heterologous POR motif, displayed notable potency for 27-hydroxylation of 5β-cholestanetriol in the absence of its native electron suppliers [391].

A suit of vectors for the expression of fungal and plant P450s as non-natural genetic fusions with various reductase isoforms have been developed. Thus, engineering of CYP51 from Saccharomyces cerevisiae to allow connection with its cognate redox partner resulted in swift 3-hydroxylanostenol demethylation [392]. Also, chimeric plant P450s CYP71B1, CYP73A and CYP76B1 were shown to work with higher overall capacity when plant reductases were permitted to act as fusion partners. Here, cinnamate 4-hydroxylation, a key reaction in phenylpropanoid biosynthesis, induces swift production of relevant secondary metabolites steering plant development, while catabolism of recalcitrant herbicides such as chlortoluron is of major importance in defense reactions [393, 394, 395, 396].

Efforts were also undertaken to evaluate exploitation of the Bacillus megaterium (CYP102A1) reductase component (BMR) as a surrogate of POR, having 35 % sequence identity with the microbial analog [397]. In this way, a series of soluble self-sufficient CYP2C chimeras, generated by gene-fused assembly of the N-terminally modified P450s with BMR via a Pro-Ser-Arg linker, displayed activities toward prototypic marker substrates comparing favorably with those reported for the wild-type enzymes [381, 398, 399]. Similar observations were made with the CYP2E1/BMR and CYP3A4/BMR constructs, though coupling levels between product formation and NADPH consumption did not exceed 8–15 % [398, 400, 401]. Furthermore, swapping of the oxidoreductase module of the P450-like self-sufficient neuronal nitric oxide synthase for BMR was found to give rise to a manipulated multi-domain construct of low stability, nevertheless displaying appreciable oxygenase activity prone to the regulatory action of the calmodulin messenger protein [402]. Finally, fusion-mediated development of a reaction host for efficient 3′-hydroxylation of 4′,7-dihydroxyisoflavone (daidzein) was carried out by cross-linking CYP105D7 via a 20 amino acid peptide to the BMR-like reductase fragment of the self-contained CYP102D1 from Streptomyces avermitilis. The engineered enzyme metabolized daidzein at a kcat/Km value 24-fold higher than that measured with CYP105D7 reconstituted with Pdx/PdR [403].

10.4.2.2 P450/Ferredoxin/Ferredoxin Reductase Fusion Enzymes

A novel type of architecture was tested for utility in simplifying the P450-dependent redox machinery. Thus, microsomal rat CYP1A1 was manipulated by gene fusion to obtain a triple adduct encompassing Fdx and FdR from plant chloroplasts. Here, the CYP1A1/Fdx/FdR order revealed to permit the most efficient oxidative turnover of 7-ethoxycoumarin and the herbicide chlortoluron [404]. Similarly, mammalian mitochondrial CYP11A1, CYP11B1 and CYP27A1 enzymes were tethered to their native accessory redox partners via the production of a series of expression cassettes. Again, arrangement of the ligated modules was recognized to have a pivotal impact on the catalytic potency of the individual constructs, with the P450/AdR/Adx congener being superior to other species. This suggested Adx to be a key factor in determining the reaction rate [405, 406, 407].

Moreover, bacterial CYP101A1 from Pseudomonas putida was fixed to its dedicated electron donors to yield a tandem linear fusion enzyme. Of note, highest NADH-promoted camphor turnover was attained with an assembly, where the PdR/Pdx duo, linked by peptides of variable length, preceded the P450 domain, though activity as such was but 30 % that of the reconstituted wild-type system [218]. In contrast to this, a novel site-specific, branched CYP101A1 fusion protein with spatially equal geometry of the three-redox-component adduct was created to minimize structural constraints. To this end, the P450 module cross-linked with PdR via a peptide, including a reactive glutamine residue and Pdx attached to a lysine-bearing tag at the C-terminus, were associated with each other by the help of transglutaminase. This product displayed tenfold higher activity compared to the simple chimera described above [408]. Using PCNA, a DNA sliding clamp, as a scaffold in the engineering of a ring-shaped heterotrimeric complex of CYP101A1 tightly juxtaposed to its attendant electron donors, catalytic activity of the resulting construct could be raised to a level two orders of magnitude higher than that of the P450 alone [409]. Notably, a thermostable system was modeled by linkage of CYP175A1 from Thermus thermophilus to a new type of FdR and Fdx, with five small amino acids being inserted as a hinge between each component to increase flexibility. The fused protein displayed full NADPH-driven reactivity toward β-carotene even at 70 °C [410].

10.4.2.3 P450/Dioxygenase Reductase-Like Fusion Enzymes

Stimulus was given by the CYP116B2 precedent to mimicking the fusion organization of the enzyme’s redox center. Thus, a plant-bacterial chimera was created by ligating the P450 domain of CYP93C1 from the soybean Glycine max to the PDR-like reductase module of the rhodococcal monooxygenase, catalyzing naringenin-to-genistein transformation at improved efficiency compared to hemoprotein mated with a usual plant reductase [395]. The same procedure was employed to engender genetically engineered merging of the FMN/Fe2S2-containing carrier moiety with the C-terminal heme unit of CYP101A1, CYP153A or CYP203A, yielding biocatalysts avidly attacking a diversity of compounds such as d-camphor, alkanes and 4-hydroxybenzoate [374, 411, 412, 413]. Also, interest focused on harnessing improved catalytic potency and broadening of substrate spectra upon fusion of the native or mutated, macrolide biosynthetic CYP107L1 protein with the PDR-type building block [414, 415]. Moreover, strategies were developed to attach the isolated heme domain of the explosive-degrading CYP177A1(XplA) via a 16-amino-acid-linker to the modified C-terminal reductase partner of CYP116B2. The artificial adduct revealed substrate specificities comparable to those of the wild-type enzyme with a Kd value for RDX docking of ~5 μM [362, 374]. It should be noted that a rare bacterial reductase has been purified from Nocardia farcinica bearing some resemblance to the molecular organization of PDR, though carrying an NADPH/FAD-binding module and an Fe4S4 cluster. Fusion of the electron donor with CYP51 gave a chimera that demethylated lanosterol at a 35-fold higher efficiency relative to the P450 unit alone [416].

10.5 Procedures to Evade Requirements for Supporting Redox Proteins and Cofactor Utilization

10.5.1 The Peroxide Shunt Pathway

The peroxide shunt serves in driving P450-catalyzed monooxygenations in the absence of an NADPH-dependent redox partner by reacting ferric hemoprotein with H2O2 or organic peroxides to generate the Fe3+-OOH intermediate, protonation of which leads to release of water and formation of the high-energy iron-oxene species [417]. In this regard, the single-component bacterial peroxygenases CYP152B1 from Sphingomonas paucimobilis, CYP152A1 from Bacillus subtilis and CYP152A2 from Clostridium acetobutylicum, primarily catalyzing α- and β-hydroxylation of long-chain fatty acids, may be ideal model systems [28, 238, 418]; here, salt bridge formation between the fatty acid’s carboxylate and an arginine located near the heme was shown to be essential to H2O2 ligation and proton delivery to initiate facile O-O bond cleavage [419]. More recently, a new member of the CYP152 family was purified from a Jeotgalicoccus species, operating in the peroxide-dependent biosynthesis of 1-alkenes via fatty acid decarboxylation [420]. Moreover, the microbial CYP107AJ1 from Streptomyces peuceticus has been ascribed to a putative peroxygenase class of P450s owing to lack of reactivity toward NADPH-driven redox partners, contrasting with the high catalytic efficiency in H2O2-supported 7-ethoxycoumarin dealkylation [421]. Similarly, human CYP2S1 was found to be resistant to reduction by POR, while mediating swift oxidative metabolism of a series of environmental carcinogens in the presence of hydrogen peroxide, cumene hydroperoxide or fatty acid hydroperoxides [26, 27]. In analogy, mammalian brain CYP2D18 was detected to support conversion of dopamine to aminochrome exclusively in a peroxygenase mode [422].

Apart from this specific behavior, P450s usually accepting reducing equivalents from a natural redox partner may, nevertheless, exploit peroxides as alternative oxygen donors in substrate biotransformations. For instance, CYP2B4 was demonstrated to utilize cumene hydroperoxide or fatty acid hydroperoxides to bring about N-oxidation of 4-chloroaniline. Albeit, turnover was found to occur at a rate not exceeding 25 % of that observed with the pyridine nucleotide-driven process [423, 424]. Notably, exchange of the enzyme’s highly conserved T302 for alanine was recognized to accelerate inactivation of the mutant through peroxide-induced denaturation of the apoprotein matrix and degradation of the heme macrocycle, pointing at a function of the threonine residue in P450 stabilization or diminution of the level of free reactive oxidant [425]. Employing CYP2D6 and CYP3A4 as probe catalysts because of their high substrate promiscuity, efficiency of the peroxygenase-like metabolic route relative to that determined by the action of natural cofactors was shown to largely rely on the type of “oxygen surrogate” employed [231, 426]. To maximize productive interactions of P450s with peroxides and minimize oxidative hemoprotein damage, substantial work was done by genetic enzyme engineering. Thus, CYP3A4 was subjected to random and site-directed mutagenesis to engender formation of a F228I/T309A variant characterized by a Vmax/Km for cumene hydroperoxide-supported 7-benzyloxyquinoline debenzylation 11-fold higher than the value observed with the wild-type enzyme. However, kcat as such only amounted to ~18 % the level measured with CYP3A4 fortified with NADPH [427]. Among bacterial P450s, three random mutants that showed improved capacity for H2O2-dependent naphthalene oxidation were generated from CYP101A1. Here, DNA sequencing revealed that amino acid substitutions C242F, R280L and E331K potentially interfered with peroxide binding [428]. Kinetic analysis of the F87A mutant of full-length CYP102A1 unveiled the modified enzyme to be a somewhat more efficient utilizer of H2O2 in medium-chain fatty acid hydroxylation compared to the parental species, where peroxide-supported activity is hardly detectable, but to shift C-H bond functionalization away from the terminal position [429]. Applying sequential rounds of random mutagenesis, peroxidative catalyst performance of the F87A-modified heme domain of CYP102A1 was drastically improved by evolution of an allelic variant carrying nine additional amino acid substitutions dispersed throughout the protein scaffold, with exception of the active-site cavity and substrate access channel [430]. Enhanced H2O2-driven peroxygenase activity was shown to extend to fatty acid substrates and styrene, although major limitations of this system are rapid suicide inactivation as the result of peroxide-mediated heme destruction and significant decrease in thermostability [430]. Here, quantum mechanical and molecular mechanical calculations allowed rational identification of key oxidizable targets, permitting replacement of the latter with less sensitive entities. In fact, the double mutant W96A/F405L gave a more stable construct [431]. Moreover, thermostabilization of the laboratory-evolved heme-domain peroxygenase variant was achieved by further directed evolution, leading to the introduction of eight new amino acid substitutions [432].

10.5.2 Photo- and Electrochemical Manipulation of the P450 System

Innovative approaches to supersede the obligatory proteinaceous redox chains in the P450 toolbox include light-induced electron transfer to the heme iron via photoactivatable mediators or direct delivery of reducing equivalents from electrodes to promote substrate metabolism [34, 433]. In this way, light-induced reductive dehalogenation of environmental pollutants such as pentachloroethane was brought about in reaction mixtures containing EDTA/proflavin and CYP101A1. Activity remained unaffected upon the addition of exogenous Pdx [434]. Great promise was also shown by the construction of hybrid CYP102A1 heme domains consisting of Ru(II)-diimine photosensitizers attached to the single cysteine residues of the K97C, Q109C, Q397C and L407C mutants, strategically positioned in close proximity to the heme. Continuous irradiation of the systems with visible light permitted hydroxylation of lauric acid with variable total turnover numbers, with the L407C variant being the most efficient catalyst despite some degradation due to oxidative damage [435]. Similarly, cadmium sulfide semiconductor nanoparticles, frequently referred to as quantum dots (QDs), have attracted interest due to their unique size-tunable properties and high photostability during the light-dependent generation of free superoxide and hydroxyl radical species [436]. Adsorption of the positively charged hexahistidine-tagged CYP152A1 on the mercaptoacetic acid-capped QD surface was recognized to yield nanohybrids of differential spatial conformation [437]. UV light-induced triggering of the hemoprotein’s peroxygenase activity was shown to cause α- and β-hydroxylation of myristic acid as well as conversion of N-acetyl-3,7-dihydroxyphenoxazine to resorufin at a rate 50 % of that found with H2O2 as the oxidant [438, 439].

Interfacing of P450s to viable amperometric devices to obtain highly efficient catalysis through direct mediator-free transfer of reducing equivalents requires modification of electrodes with agents that facilitate electron flow, prevent protein denaturation and cause appropriate orientation of the enzymes. To attain this goal, different types of bioelectrocatalysts have been developed [440]. Thus, riboflavin-bearing CYP1A2, CYP2B4 and CYP11A1 enzymes entrapped in a phospholipid vesicular system were cross-linked via glutaraldehyde to screen-printed (SP) thick film rhodium-graphite working electrodes, poised at −500 mV vs. Ag/AgCl reference electrodes. Rates of biosensor-driven p-hydroxylation of aniline, N-demethylation of aminopyrine and cholesterol side-chain cleavage were close to those obtained with NAD(P)H as the electron source [441]. Alternatively, CYP2B4 was adsorbed onto SP electrodes coated with colloidal gold nanoparticles stabilized with didodecyldimethylammonium bromide (DDAB) in the presence of the Nafion ionomer to improve film permeability. The construct adequately mediated benzphetamine N-dealkylation [442]. Modifying the immobilization scheme, studies were carried out with monomerized CYP2B4 incorporated into thin layers of nonionic detergent and montmorillonite, a member of the mineral group of clays, on glassy carbon (GC) electrodes. Here, kcat for aminopyrine turnover was shown to be comparable to the value of the microsomal system [443]. Moreover, a biocompatible film containing colloidal gold nanoparticles and chitosan was used to encapsulate CYP2B6 on GC sensors. Product analysis confirmed C-hydroxylation and heteroatom release from bupropion, lidocaine and cyclophosphamide to be the main pathways of drug oxidation [444]. Studies were also conducted with carbon cloth (CC) electrodes coated by immersion into DDAB dispersions embedding bacterial CYP101A1. Electrolyses performed under aerobic conditions in the presence of styrene and cis-β-methylstyrene as the probe substrates revealed styrene oxide and trans-β-methylstyrene oxide to be the major products resulting from oxidative attack by the P450, while some byproducts were speculated to rather arise from H2O2-driven reactions [445].

Substantial progress was achieved by construction of enzyme films of predesigned architecture via layer-by-layer self-assembly of hemoproteins and oppositely charged polyions on the surface of electrodes. Applying this regimen, CC sensors elaborated by casting CYP1A2/poly(styrenesulfonate) (PSS) microemulsions onto the solid supporters displayed good electrocatalytic performance of O2 reduction to hydrogen peroxide, mediating epoxidation of styrene faster than CYP101A1 [446].

Electrochemical exploration was extended to assembly of enzyme films on the surface of derivatized gold electrodes by alternate adsorption of a P450 layer on top of a poly(diallyldimethyl-ammonium) (PDDA) layer. In this way, immobilization of CYP2E1 and CYP3A4 resulted in sensor devices mediating oxidative turnover of p-nitrophenol and midazolam, respectively, at fairly low catalytic rates [447, 448]. Here, covalent enzyme linkage to gold electrodes via flexible spacer molecules, bearing both thiol and disulfide groups as well as anchors to the proteins, was shown to increase metabolic efficiency. Making use of this strategy, the exposed C261 and C268 residues of CYP2E1 were intimately connected with cystamine-maleimide on gold biosensors. This procedure stimulated conversion of p-nitrophenol to p-nitrocatechol by a factor of 22 relative to the Au/PDDA array [447]. Similarly, human CYP2C9 was bonded to a gold electrode by the aid of its N-terminal lysine fixed to an 11-mercaptoundecanoic acid and octanethiol self-assembled monolayer. Electron transfer was calculated to proceed at a rate ranging from 6 to 31 s−1, with warfarin being metabolized to the 7-hydroxy derivative at an apparent Km of 3 μM [449].

10.6 Conclusions and Future Prospects

The present review focuses on the description of electron transfer events with emphasis on topological and functional features in the P450-dependent redox chain to gain a more detailed, structure-based insight into fundamental molecular principles steering donor-acceptor interactions. Improved comprehension may permit engineering to introduce more efficient electrochemical properties into the system such as facilitated redox partner association and intermolecular electron flow [200, 303], but equally-well may pave the way for the development of technologically viable hemoprotein species for extensive exploitation as versatile biocatalysts [35]. Here, directed evolution and DNA shuffling may be useful in the sculpturing of self-sufficient fusion proteins for preselected metabolic implementation [376, 395, 404] or in the development of peroxygenase-like P450s with upgraded resistance toward oxidative destruction [430, 431] to obviate the tedious reconstitution procedure. Also, artificial photo- and electrocatalytic devices might help avoid costly NAD(P)H utilization [434, 442].

Despite conspicuous biotechnological advances [450], industrial large-scale production of fine chemicals is presently limited to a fairly low number of processes making preferential use of microbial whole-cell catalysts harboring recombinant P450s co-expressed with an appropriate electron donor [451]. Relevant examples include hydrocortisone production via P450lun-mediated 11β-hydroxylation of 11-deoxycortisol in a fungal bioreactor [452] or manufacture of the cholesterol-lowering drug pravastatin by CYP105A3-driven attack on compactin, employing the Streptomyces sp. Y-110 as the host [453]. Moreover, CYP71AV1-promoted three-step oxidation of amorphadiene to artemisinic acid, the immediate precursor of the antimalarial drug artemisinin, permitted industrial scale-up due to high productivity of the engineered Saccharomyces cerevisiae factory [454]. Similarly, long-chain α,ω-dicarboxylic acids, widely used as raw materials for the synthesis of products such as perfumes, hot-melting adhesives, engineering plastics or high quality lubricants, have been generated on a commercial scale from n-alkanes by fungal fermentation catalyzed by Candida tropicalis, housing CYP52A1 in conjunction with POR as the redox machinery [455].

Specialized exploitation of manipulated hemoproteins was recognized to be of high interest in gene-directed enzyme prodrug therapy (GDEPT) of cancer. Here, introduction of tumor-selective retroviral vectors, encoding P450s characterized by high metabolic potency in the reductase-supported intratumoral conversion of anticarcinogenic compounds such as cyclophosphamide or ifosfamide to their active intermediates, displayed a substantial therapeutic progress [456]. This has given an impetus to improvement of reactivity of CYP2B enzymes toward the oxazaphosphorines [457, 458]. In this regard, creation of the 114V/477W double mutant of human CYP2B6 increased the catalytic efficiency of cyclophosphamide oxidation by a factor of 4 [459]. Also, there is clear opportunity to expedite therapeutic efficacy by utilization of the fusion gene of the self-sufficient CYP2B6/POR chimera for infection of tumor cells [460]. The current advances lend confidence that this novel strategy may be promoted by the development of more sophisticated vector and promotor systems.

The engineered P450 redox machinery may also be exploited in phyto- and bioremediation processes. Thus, expression in tobacco plants of human CYP1A proteins or CYP76B1 from Helianthus tuberosus as hybrid enzymes fused with POR increased herbicide resistance toward a series of phenylureas such as chlortoluron due to swift detoxification [379, 396]. Similarly, transduction of rice plants with human CYP2B6 or CYP2C19 enhanced the ability to remove atrazine and metolachlor herbicides from soil [461]. Interest has also arisen in transgenic Arabidopsis plants producing the bizarre fusion protein CYP177A1(XplA) for targeted degradation of the widespread military explosive RDX, a priority pollutant contaminating liquid culture and soil leachate [462]. One factor complicating introduction of such technologies for environmental clean-up may be concerns about field application of genetically modified organisms, possibly entailing certain risks. Nevertheless, remediation of the biotope needs a robust catalytic apparatus capable of killing off hazardous anthropogenic toxicants via more flexible, pollutant-specific oxyfunctionalization. Here, polycyclic aromatic hydrocarbons such as phenanthrene, naphthalene, fluorene or benzo[a]pyrene proved to be targets for optimized biotransformation by chimeric CYP1A1/POR, mutated CYP102A1, fused CYP116B3 or modified CYP5136A3, expressed in microbial recombinant cells [371, 463, 464, 465]. However, inoculation and efficient maintenance of the population density of the engineered microbial biomass in terrestrial habitats still need improvement [466].

Finally, exploitation of P450-based electroanalytical techniques may become of increasing interest to enable more practical applications. Given appreciable sensitivity and recognition selectivity, miniaturized amperometric biosensors might be utilized for the determination of compounds important in pharmaceutical industry, clinical practice and environmental monitoring [467, 468]. Also, microfluidic devices were developed to improve analytical performance by decreasing analysis time and increasing reliability through automation [440]. This may foster high-throughput screening during the search for structural features of dynamic molecules having potential for therapeutic implementation [469].

Collectively, catalytic versatility, no doubt, adds the P450 redox system to the enzymatic armory for large-scale exploitation in a vast array of biotechnological areas. Despite huge progress in recent years, members of the hemoprotein family are, nevertheless, thought of as relatively fragile biocatalysts prone to spontaneous structural disruption or rapid inactivation at temperatures >40 °C [430]. Hence, future engineering strategies will have to focus on erasure of these shortcomings and evolution of novel activities to allow widespread application in metabolic processes.

Notes

Reviews

Abbreviations: AdR NADPH-adrenodoxin reductase, Adx adrenodoxin, Arx [2Fe-2S]-type ferredoxin, b5 cytochrome b5, BMR Bacillus megaterium (CYP102A1) reductase component, CC carbon cloth electrode, CYP or P450 cytochrome P450, FdR NAD(P)H-ferredoxin reductase, Fdx ferredoxin, GC glassy carbon electrode, PCNA proliferating cell nuclear antigen, PdR NADH-putidaredoxin reductase, PDR phthalate dioxygenase reductase, Pdx putidaredoxin, POR NADPH-P450 oxidoreductase, Pux palustrisredoxin, r.m.s. root mean square deviation, SP screen-printed electrode.

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© Springer International Publishing Switzerland 2015

Authors and Affiliations

  1. 1.Walther-Straub-Institut für Pharmakologie und Toxikologie der LMUMünchenGermany

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