Encyclopedia of Biophysics

Living Edition
| Editors: Gordon Roberts, Anthony Watts, European Biophysical Societies

Bacterial Globins

  • Robert K. PooleEmail author
  • Mark Shepherd
Living reference work entry
DOI: https://doi.org/10.1007/978-3-642-35943-9_34-1
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Keywords

Bacterial Globin Single-domain Globins Flavohemoglobin Globin Fold Myoglobin Fold 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Synonyms

Definitions

Bacterial globins are proteins possessing the classical globin fold and the highly conserved active site residues required for ligand-binding function. Functions include binding of, or reaction with, oxygen, nitric oxide, carbon monoxide, or other ligands. Bacterial globins are classified into three major groups – myoglobin-like globins (two- or single-domain proteins), sensor globins, and truncated globins. NO detoxification is a well-characterized function of some, but there is inadequate functional data.

Basic Characteristics

Globins are an ancient superfamily of diverse proteins (Vinogradov et al. 2006). The first report of a microbial globin was in yeast over half a century ago, but, in the past 20 years, increasing molecular studies have revealed details of their structures and functions and the regulation of their biosynthesis. There has been a dramatic increase in the understanding of bacterial globins and the links with bacterial respiration and physiology, pathogenesis, and biotechnological opportunities, fueled by the recognition that a major role of certain microbial globins is protection from nitric oxide (NO) (Poole and Hughes 2000; Wu et al. 2003).

The subunits of all hemoglobins consist of a polypeptide chain with 6–8 α-helical segments that fold around a heme group (Fig. 1a) (Shepherd et al. 2010). The helices constituting the globin fold are labeled A to H in sequence order, and the various topological positions within each helix are also numbered sequentially. The fifth coordination site of the central iron atom of the heme is occupied by the imidazole ring from a histidine residue on the protein. This proximal ligand (F8 in Fig. 1b) is the residue at position 8 in helix F and so may be written HisF8 or His-xy (where xy is the number of the residue in the entire linear globin primary structure, not in a particular helix). The distal ligand-binding pocket occupies the site diametrically opposite the proximal histidine. This sixth coordination site is available to bind oxygen or another ligand. It is typically constructed from the B, E, and part of the G helices. The identity of the B10 residue on the B helix and the E7 and E11 residues are important for the regulation of ligand binding and discrimination. In mammalian globins, the E7 position is almost invariably occupied by a histidine. The HisE7 is believed to stabilize the heme-bound dioxygen by H-bonding to it; on the other hand, the B10 and E11 positions are typically occupied by hydrophobic residues to ensure that the chemical integrity of the heme-bound dioxygen is preserved. In contrast, the distal residues in the hemeproteins performing oxygen chemistry, such as peroxidases and oxidases, are much more polar. For example, the B10 and E7 residues in the single-domain globin (Cgb) from Campylobacter jejuni are occupied by tyrosine and glutamine residues, respectively (Fig. 1b). The polar nature of the distal heme environment in these proteins plays an important role in activating the O-O bond of the heme-bound ligand molecules. The distal residues of microbial hemoglobins are also much more polar than those of mammalian globins, suggesting that the structures of these hemoglobins are tailored to perform functions other than oxygen transport (Lu et al. 2008).
Fig. 1

Structural features of the single-domain globin from C. jejuni, Cgb. (a) Backbone topology of Cgb. A Cα chain tracing of Cgb with heme cofactor (black). Helices/regions are labeled according to conventional globin nomenclature. (b) Structure of the distal pocket of Cgb (Shepherd et al. 2010)

Globin function is typically defined by reactivity toward small ligands, such as O2, CO, and NO, which bind to the heme distal site. In general, hemoglobins displaying a moderate oxygen affinity are involved in O2 transport or sequestration, whereas those exhibiting high oxygen affinities appear to be involved in oxygen chemistry. To perform these functions, oxygen affinity is tightly regulated, and the structural basis underlying O2 affinity has been extensively studied (Capece et al. 2006). O2 affinity is modulated by the characteristics of both the heme proximal and distal environments. Oxygen binding is determined by ligand release and association kinetics. The ligand release process can be further divided into two main steps. First, the iron-ligand bond must be broken, and, second, the ligand must diffuse into the bulk solvent through the protein matrix. In most proteins, the observed dissociation rate is determined mainly by the strength of the protein-ligand interactions, or ligand migration. Depending on the H-bonding network between heme-bound O2 and amino acid residues located in the distal site of the heme, oxygen dissociation rates (koff) can vary by more than five orders of magnitude. However, ligand association rates (kon) are usually determined by the presence of tunnels that facilitate ligand migration from the solvent toward the heme active site.

Nomenclature and Classification

The description of globins has long been confused, since the basic globin fold described above is found in a bewildering range of structurally and functionally distinct structures. However, in a bioinformatics survey of putative globins in over 2200 bacterial and some 140 archaeal genomes, over half the bacterial genomes contain genes encoding globins. These have been classified into three families: the M (myoglobin-like) and S (sensor) families, all exhibiting the canonical 3/3 myoglobin fold, and the T family (truncated myoglobin fold) (Vinogradov et al. 2013). The M family comprises two subfamilies, flavohemoglobins (FHbs) and single-domain globins (SDgbs). The S family encompasses chimeric globin-coupled sensors (GCSs), single-domain Pgbs (protoglobins), and SSDgbs (sensor single-domain globins), while the T family comprises three classes TrHb1s, TrHb2s, and TrHb3s, all characterized by the abbreviated 2/2 myoglobin fold. The smallest globin-bearing genomes are the streamlined genomes (~1.3 Mbp) of certain Alphaproteobacteria and the slightly larger (ca. 1.7 Mbp) genomes of Aquificae. The smallest genome with members of all three families is the 2.3 Mbp genome of the extremophile Methylacidiphilum infernorum (Verrucomicrobia). Of the 147 possible combinations of the eight globin subfamilies, 83 are observed. Although binary combinations are infrequent and ternary combinations are rare, the FHb + TrHb2 combination is the most commonly observed. Many functions have been proposed for bacterial globins, but in only a few cases is there unequivocal physiological or genetic evidence. These cases include nitric oxide detoxification via the NO dioxygenase or denitrosylase activities of M-class globins and the sensing of oxygen concentration in environmental niches. Here only selected examples are described.

Flavohemoglobins

Members of the best-understood class, the flavohemoglobins, are distinguished by the presence of an N-terminal globin domain (a three-on-three α-helical fold similar to myoglobin) with an additional C-terminal domain with binding sites for flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]. Widely distributed in bacteria and lower eukaryotes, flavohemoglobins confer protection from NO and nitrosative stresses by direct consumption of NO (Poole and Hughes 2000). They are critical for pathogenicity in some species; for example, E. coli and Salmonella mutants lacking Hmp, the best studied such protein, are compromised for survival in mouse and human macrophages. In the plant pathogen Erwinia chrysanthemi, HmpX not only protects against nitrosative stress but also attenuates host hypersensitive reaction during infection by intercepting NO produced by the plant for the execution of the hypersensitive cell death program. Flavohemoglobins appear to have no direct role in the reductive metabolism of oxygen or other gaseous ligands and have not been reported in higher animals. Flavohemoglobins oxidize NAD(P)H and transfer an electron to the N-terminal heme domain via a non-covalently bound FAD in the reductase (or FNR, ferredoxin-NADP reductase-like) domain. Reduced heme catalyzes the reaction between NO and O2 generating nitrate (NO3); that is, Hmp acts as a NO-detoxifying enzyme. There remains controversy over the reaction mechanism at the heme: either NO (denitrosylase mechanism) or O2 (dioxygenase mechanism) has been claimed to bind first to the heme.

In accordance with the role of Hmp in limiting NO-related toxicity, expression of the protein occurs only when NO is present in the cell environment. Indeed, hmp gene expression is tightly regulated at the transcriptional level by NO-responsive transcription factors, notably NsrR and Fnr (Spiro 2007). Constitutive Hmp expression and function in the absence of NO generate oxidative stress by virtue of oxygen reduction by the heme to superoxide anion.

Single-Domain “Myoglobin-Like” Hemoglobins

The second class of bacterial globin resembles mammalian myoglobin in having the canonical 3/3 fold but no additional domain. This class is typified by the globin of Vitreoscilla (named Vgb, VtHb, or Vhb), an obligately aerobic bacterium that grows in low-oxygen environments. This globin was the first bacterial hemoglobin to be crystallized, and the three-dimensional structure (of the ferric homodimer) conforms to the classical globin fold. However, the region following the C helix is disordered, residues E7-E10 do not adopt the usual α-helical conformation, and GlnE7 is located out of the heme pocket and does not appear to stabilize the heme iron-bound dioxygen through hydrogen bonding. The three-dimensional structures of the thiocyanate and imidazole derivatives of recombinant ferric Vgb have also been determined (Giangiacomo et al. 2001). This protein has been implicated in redox chemistry and NO detoxification in vivo, but the mechanism by which the protein is re-reduced after a catalytic cycle is obscure. The disordered CD region (i.e., the vicinity of the C and D helices) in the crystal structure of Vgb is a potential site of interaction with the putative FAD/NADH reductase partner. Considerable interest has been directed at Vgb because of its possible role in facilitating oxygen transport and metabolism. Indeed, Vgb is upregulated under microaerobic growth conditions and shows promise for biotechnological application by enhancing product formation under oxygen limitation in heterologous hosts (Frey et al. 2011).

A more comprehensive molecular genetic view of bacterial non-flavohemoglobins is afforded by the microaerophilic, foodborne, pathogenic bacterium Campylobacter jejuni, which is exposed to NO and other nitrosating species during host infection. This single-domain globin, Cgb, is dramatically upregulated by the transcription factor NssR in response to nitrosative stress (Elvers et al. 2005). Cgb has been shown to detoxify NO and possess a peroxidase-like heme-binding cleft. In marked contrast to Vitreoscilla Vgb, there is no evidence to date that Cgb functions in oxygen delivery. Cgb can provide an electronic “push” from the proximal ligand and an electronic “pull” from the distal binding pocket, creating a favorable environment for the isomerization of a putative peroxynitrite intermediate in the NO dioxygenase reaction (Shepherd et al. 2010).

Sensor Globins

The term globin-coupled sensor is used to describe chimeric proteins in which a C-terminal domain shows clear homology with known signaling domains (like the bacterial chemotaxis proteins) but the N-terminus possesses only a weak (10%) similarity to the globin fold (Gilles-Gonzalez and Gonzalez 2005). In most cases, the functions of sensor proteins have not been unequivocally proved, but a heme-based aerotactic transducer (HemAT-Bs) has been identified in Bacillus subtilis (Hou et al. 2000; Zhang et al. 2005).

Truncated Hemoglobins

The third major globin class comprises the truncated globins, which are the most recently discovered and appear widely distributed in bacteria, microbial eukaryotes, and plants. Instead of the classical 3-over-3 α-helical sandwich motif adopted by single-domain globins (including the flavohemoglobins) and by the globin-coupled sensors (Table 1), trHbs adopt a 2-over-2 α-helical structure and are typically 20 residues shorter than 3-over-3 globins. Sequence analysis of more than 200 trHbs indicates that they can be divided into three groups: 1, 2, and 3 (sometimes referred as N, O, and P, respectively) (Wittenberg et al. 2002). Most studies on trHbs have focused on trHb groups 1 and 2, although the type-3 trHb from the foodborne bacterial pathogen Campylobacter jejuni, Ctb, has been structurally and kinetically characterized (Lu et al. 2007).
Table 1

A proposed global nomenclature for globins

Family name

M (myoglobin-like globins)

S (globin-coupled sensors)

T (truncated globins)

Fold (of globin domain)

3/3

3/3

2/2

Bacterial subfamily name

FHb (flavohemoglobins)

SDgbs (single-domain globins related to FHbs, or “non-flavohemoglobins”)

CGS (chimeric globin-coupled sensors)

Pgbs (protoglobins)

SSDgbs (sensor single-domain globins)

TrHb1 = N

TrHb2 = O

TrHb3 = P

Bacterial and archaeal examples

Hmp (E. coli)

FHb (Ralstonia eutropha)

Cgb (Campylobacter jejuni)

Vhb (Vitreoscilla)

HemAT (aerotactic bacteria)

MaPgb (Methanosarcina acetivorans)

Thermus thermophilus, Methylacidiphilum infernorum

trHbN

(Mycobacterium tuberculosis)

trHbO

(Mycobacterium tuberculosis)

Ctb (Campylobacter jejuni)

Function(s)

NO dioxygenase

NO detoxification

Oxygen-responsive (aerotaxis) transducer

Unknown

Unknown

NO detoxification

Unknown

Unknown

Estimated number of sequences

>530

>220

>420

>40

>35

>240

>620

>330

Mycobacterium tuberculosis contains two trHb proteins (Table 1) (Davidge and Dikshit 2013). Oxygen-binding studies of trHb I from Mycobacterium tuberculosis (Mt-trHbN), reported to be an NO dioxygenase, show an unusually high O2 affinity (Kd = 2.3 nM), allowing oxygen binding even at low O2 concentrations. The high oxygen affinity of Mt-trHbN is achieved by the presence of two polar residues in the distal site, TyrB10 and GlnE11, which form H-bonds with the heme-bound O2 (Boron et al. 2015). Another trHb from Mycobacterium tuberculosis, Mt-trHbO (a trHb II), also displays a high O2 affinity (Kd ~11 nM). Mt-trHbO contains three polar residues in the distal cavity, TyrB10, TyrCD1, and TrpG8, which can donate H-bonds to the bound O2 (Davidge and Dikshit 2013). The mechanisms for the proposed functions are not understood in detail.

Like M. tuberculosis, C. jejuni contains two globins, Cgb and Ctb (Tinajero-Trejo and Shepherd 2013), and the latter belongs to trHb group III. When the structural gene, ctb, is mutated, the bacterium does not show extra sensitivity to nitrosative stress, but, when grown under conditions of high aeration, the bacterium exhibits lowered respiration rates, suggesting a role for Ctb in modulating intracellular O2 flux. The role of this protein in bacterial physiology remains obscure. Ctb contains three polar residues in the distal cavity, TyrB10, HisE7, and TrpG8; it is thought that HisE7 regulates ligand entry, but the mechanism by which the three distal polar residues control oxygen reactivity in Ctb remains unclear (Lu et al. 2008, 2007).

Cross-References

References

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Copyright information

© European Biophysical Societies' Association (EBSA) 2018

Authors and Affiliations

  1. 1.Department of Molecular Biology and BiotechnologyThe University of SheffieldSheffieldUK
  2. 2.School of BiosciencesUniversity of KentCanterburyUK