Identification of Cys94 as the distal ligand to the Fe(III) heme in the transcriptional regulator RcoM-2 from Burkholderia xenovorans
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The CO-responsive transcriptional regulator RcoM from Burkholderia xenovorans (BxRcoM) was recently identified as a Cys(thiolate)-ligated heme protein that undergoes a redox-mediated ligand switch; however, the Cys bound to the Fe(III) heme was not identified. To that end, we generated and purified three Cys-to-Ser variants of BxRcoM-2—C94S, C127S, and C130S—and examined their spectroscopic properties in order to identify the native Cys(thiolate) ligand. Electronic absorption, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopies demonstrate that the C127S and C130S variants, like wild-type BxRcoM-2, bind a six-coordinate low-spin Fe(III) heme using a Cys/His ligation motif. In contrast, electronic absorption and resonance Raman spectra of the C94S variant are most consistent with a mixture of five-coordinate high-spin and six-coordinate low-spin Fe(III) heme, neither of which are ligated by a Cys(thiolate) ligand. The EPR spectrum of C94S is dominated by a large, axial high-spin Fe(III) signal, confirming that the native ligation motif is not maintained in this variant. Together, these data reveal that Cys94 is the distal Fe(III) heme ligand in BxRcoM-2; by sequence alignment, Cys94 is also implicated as the distal Fe(III) heme ligand in BxRcoM-1, another homologue found in the same organism.
KeywordsHeme Electron paramagnetic resonance Resonance Raman spectroscopy Site-directed mutagenesis Transcription
Acetobacter xylinum phosphodiesterase A1
Brain and muscle Arnt-like protein 1
Burkholderia xenovorans regulator of CO metabolism
Escherichia coli direct oxygen sensor
Electron paramagnetic resonance
Heme-regulated α-subunit of eukaryotic translational initiation factor 2 kinase
Ligand-to-metal charge transfer
Methanobacterium thermoautotrophicum direct oxygen sensor
Neuronal PAS domain protein 2
Regulator of CO metabolism
Rhodospirillum rubrum CooA
Small, gaseous diatomic molecules, such as O2, NO, and CO, are important signaling molecules in biological systems; one method nature employs to sense these gases is interaction of the diatomic molecule with a heme cofactor [1, 2, 3, 4, 5, 6]. Dioxygen binding to heme in myoglobin and hemoglobin is a well-known paradigm. Examples of heme-based O2 sensor proteins, in which the dioxygen-binding heme domain regulates function, include Rhizobium meliloti FixL (an O2-sensing heme protein associated with N2 fixation gene regulation) , Bacillus subtilis heme-based aerotactic transducer , and Escherichia coli direct oxygen sensor (EcDOS) . NO is produced endogenously by nitric oxide synthase; at physiological levels, NO plays a role in both neurotransmission and vascular regulation . Examples of heme-containing NO sensors include the bacterial heme nitric oxide/oxygen binding domain proteins , mammalian soluble guanylyl cyclase , heme-regulated α-subunit of eukaryotic translational initiation factor 2 kinase (HRI) , and the putative NO sensors Drosophila melanogaster hormone receptor 51 , heme-containing D. melanogaster nuclear receptor E75 , and Pseudomonas aeruginosa dissimilative nitrate respiration regulator . CO is also believed to function as a neurotransmitter in mammals, where it is endogenously produced by heme oxygenase . Examples of CO sensors include mammalian neuronal PAS domain protein 2 (NPAS2)  (the PAS domain is a domain structure named for the proteins Period, aryl hydrocarbon receptor nuclear translocator, and Simple-minded) and bacterial Rhodospirillum rubrum CooA (RrCooA), a heme-containing CO-sensing transcription factor [19, 20], as well as the putative CO sensors D. melanogaster E75  and D. melanogaster hormone receptor 51 .
Recently, a new prokaryotic transcriptional regulator of CO metabolism (RcoM) was isolated from the aerobic, polychlorinated biphenyl degrading bacterium Burkholderia xenovorans (LB400) [21, 22]. Two homologous proteins, B. xenovorans RcoM (BxRcoM)-1 and BxRcoM-2, were identified in this bacterium and share 93 % sequence similarity. These proteins contain an N-terminal PAS domain and a C-terminal LytTR domain, a DNA-binding domain from the AlgR/AgrA/LytR family. PAS domains frequently function as sensors of environmental signals through the binding of small molecules and may bind various cofactors . LytTR domains are DNA-binding domains that commonly function within transcriptional regulators and may be found in conjunction with PAS sensor domains . In BxRcoM-1 and BxRcoM-2, the N-terminal PAS domain binds a b-type heme cofactor that controls CO-dependent DNA binding (Kerby et al., unpublished).
Studies of BxRcoM-1 and BxRcoM-2 revealed the ligation and oxidation state changes that occur at the RcoM heme. The BxRcoM-1 and BxRcoM-2 homologues were first isolated under aerobic conditions, yet they each contained a six-coordinate Fe(II)CO-bound heme . Aerobic photolysis of the Fe(II)CO species resulted in conversion to a six-coordinate, low-spin Fe(III) heme. Site-directed mutagenesis of BxRcoM-1 identified His74 as the proximal ligand, and spectral characterization of Fe(III)BxRcoM-2 definitively demonstrated that the protein bears a cysteine(thiolate) ligand trans to a neutral ligand, presumably the same His74. BxRcoM-2 was shown to undergo a redox-mediated ligand switch in which the cysteine(thiolate) ligand is lost upon heme reduction. The spectral signatures of Fe(II)BxRcoM-2 were consistent with the presence of methionine trans to His74; mutagenesis data suggested that the distal Fe(II) ligand for BxRcoM-1, and by analogy BxRcoM-2, is Met104 [21, 25]. Methionine, a weaker ligand than histidine, may be more easily replaced by CO; consistent with this understanding, resonance Raman data of the heme–CO adduct in BxRcoM-2 were indicative of CO binding trans to the proximal His74 . Thus, the proximal ligand His74 remains constant during redox- and CO-binding events, whereas the distal ligand is exchanged in order to allosterically modulate BxRcoM DNA-binding behavior to initiate transcription. Although the mutagenesis studies were on BxRcoM-1 and the spectroscopic studies were on BxRcoM-2, the presence of His74 and Met104 in the N-terminal PAS domains of both proteins implies that the same amino acids are the Fe(II) ligands in these two closely related homologues.
Herein, we present identification of Cys94 as the cysteine(thiolate) ligand to the Fe(III) heme in BxRcoM-2. Because there were only three cysteines, we chose to isolate variants in which each of the three was altered; surprisingly, the evidence unequivocally identifies Cys94 as the thiolate ligand. Spectroscopic signatures of the three BxRcoM-2 variants—C94S, C127S, and C130S—were studied using electronic absorption, resonance Raman, and electron paramagnetic resonance (EPR) spectroscopies. The Fe(III) C94S variant exhibits differences in its spectroscopic features from those of wild-type Fe(III)BxRcoM-2 and the other variants, and the spectral signatures of Fe(III) C94S are inconsistent with coordination of a cysteine(thiolate) ligand to the Fe(III) heme.
Materials and methods
Glycerol (greater than 99.5 % purity) and all chemicals used in buffer preparation (99.5 % purity or better) were purchased from Sigma-Aldrich and used as received. Certified ACS grade potassium ferricyanide—K3[Fe(CN)6]—was purchased from Sigma-Aldrich and used as received. Sodium dithionite (Na2S2O4; 85 % purity) was purchased from Fluka and stored under argon gas at 253 K until it was used. A CO gas (99.5 % purity) cylinder was obtained from AGA.
Mutagenesis, isolation, and purification of BxRcoM-2 variants
Variant proteins were generated by mutagenesis of the cloned BxRcoM-2  according to the QuikChange protocol [Agilent Technologies (Stratagene), Santa Clara, CA, USA]. Sequences of all constructs were verified with BigDye v. 3.1 reaction chemistry [Life Technologies (Applied Biosystems), Carlsbad, CA, USA] and subsequent analysis by the University of Wisconsin–Madison Biotechnology Center DNA sequencing facility.
Isolation and purification of the BxRcoM-2 variants was similar to that described in our previous reports [21, 25]. Briefly, E. coli VJS6737  bearing the pEXT20 expression vector  for the appropriate variant was cultivated in rich medium supplemented with ferric citrate. Isopropyl β-d-thiogalactopyranoside (7 μM) was used to induce expression for 18–20 h at 301 K. Cells were pelleted by centrifugation (10,000g, 10 min), resuspended in lysis buffer [50 mM 3-(N-morpholino)propanesulfonic acid (MOPS), pH 7.5, 500 mM KCl, and 0.5 mM dithiothreitol], and lysed by passage through a French press. A nickel nitriloacetic acid column (Qiagen, 8-mL resin volume) was preequilibrated with 10 mM imidazole, 50 mM MOPS, pH 7.5, 500 mM KCl. The cell supernatant (25 mL) was applied slowly, and the column was washed successively with 10 mM imidazole (two column volumes) and 50 mM imidazole (one column volume), in the same buffer. Finally, protein was eluted with 220 mM imidazole. The protein was precipitated at 55 %-saturated (NH4)2SO4 (v/v) with incubation on ice for 30 min and brief centrifugation. The protein pellet was dissolved in 500 μL 25 mM MOPS, pH 7.4 and 500 mM KCl, applied to a buffer-equilibrated Sephadex G-25 column, and eluted in the same buffer. The desalted protein was stored at 193 K. Protein concentrations were determined using the bicinchoninic acid method (Pierce, Rockford, IL, USA); sodium dodecyl sulfate polyacrylamide gel electrophoresis verified protein purity was greater than 90 %; heme content was determined using the pyridine hemochromogen assay .
Electronic absorption spectroscopy
Electronic absorption spectra were recorded with a double-beam Varian Cary 4 Bio spectrophotometer set to a spectral bandwidth of 0.5 nm. Spectra were acquired at room temperature for samples of protein prepared, as indicated in the figure legends, in either 25 mM MOPS buffer, pH 7.4, or 25 mM 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (EPPS) buffer, pH 8.0, with 500 mM KCl. Oxygen was removed from anaerobic samples by flowing argon gas through the headspace of a septum-sealed cuvette for at least 10 min. Reduction of Fe(III) protein samples was accomplished either by adding an anaerobically prepared stock solution of sodium dithionite to achieve a final sample concentration of 1–5 mM or by adding a few solid crystals of sodium dithionite to the protein sample solution with argon gas flowing in the cuvette headspace. The Fe(II)CO adducts were prepared by the injection of CO via a gastight syringe into the headspace of a septum-sealed cuvette containing the Fe(III) or as-isolated protein, followed by gentle agitation of the sample and addition of the dithionite reductant. Final protein and heme concentrations ranged from 7 to 10 μM, and 100–200 μL of CO gas was injected, as appropriate, to give complete conversion to the Fe(II)CO adduct.
Reoxidation of the Fe(II)BxRcoM-2 variants
Protein that was present in the Fe(II) state to a significant extent after purification was reoxidized using potassium ferricyanide. A stock solution of potassium ferricyanide (500 μL, 25 mM) was prepared by dissolving 4–5 mg of solid potassium ferricyanide in 500 μL of 25 mM MOPS, pH 7.4, with 500 mM KCl. This stock solution was added to the protein sample to a final concentration of 1–2 mM. The mixture was allowed to react for 20–30 min. The protein solution was loaded onto a YM-30 Amicon Ultra spin concentrator (Millipore) and washed four times by concentration and dilution into fresh MOPS buffer (25 mM pH 7.4, 500 mM KCl) using a tabletop centrifuge (relative centrifugal force 15,000g) to remove excess potassium ferricyanide. Final heme and protein concentrations were determined using the pyridine hemochromogen assay  and the bicinchoninic acid assay (Pierce, Rockford, IL, USA), respectively.
X-band EPR spectra were collected with a Bruker ELEXSYS E500 instrument equipped with an Oxford Instruments ESR 900 continuous-flow liquid helium cryostat and an Oxford Instruments ITC4 temperature controller maintained at 10 K. The microwave frequency was monitored using an EIP model 625A continuous-wave microwave frequency counter. Protein samples were prepared, as indicated in the figure legends, in either 50 mM borate, pH 8.0, or 25 mM MOPS, pH 7.4, with 500 mM KCl. Samples were transferred to a quartz EPR tube via small-bore tubing (connected to a gastight syringe) and frozen in liquid nitrogen. Samples of approximately 150 μL had a final concentration of 80–250 μM heme. For all samples, scans from 0 to 10,000 G revealed no signals other than those reported.
Resonance Raman spectroscopy
Resonance Raman spectra were obtained with an excitation wavelength of 413.1 nm from a Coherent I-302C Kr+ laser. Low incident laser powers of less than 20 mW were focused with a cylindrical lens onto the sample. The frozen protein samples, prepared as described for EPR spectroscopy (vide supra), were placed in a quartz dewar and maintained at 77 K in liquid nitrogen to reduce local heating. Light was scattered in an approximately 135° geometry (relative to the sample) and dispersed by an Acton Research triple monochromator with gratings of 2,400 grooves per millimeter. Scattered light was detected using a liquid-nitrogen-cooled CCD camera (Princeton Instruments) under computer control using the SpectraSense software package. Peak positions were calibrated relative to the ice peak at 228 cm−1 or a Na2SO4 peak at 981 cm−1. Windows centered at 650, 1,250, and 1,850 cm−1 were overlaid for a total frequency range of 150–2,250 cm−1. IGOR Pro version 6.0 (Wavemetrics) was used to import and process all spectral data. Spectral deconvolutions were achieved using IGOR Pro version 6.0. Peaks were generated by direct combination of a series of Gaussian curves; the amplitudes and frequencies of the Gaussian curves were manually adjusted until the residuals between the native peak and the fitted peak were minimized. Major vibrational modes were assigned on the basis of comparison with those of other heme proteins and the work of Kitagawa and Spiro [29, 30, 31, 32, 33].
Characterization of C127S and C130S BxRcoM-2
Comparison of electronic absorption peak positions (nm) for the CxxS Burkholderia xenovorans regulator of CO metabolism 2 (BxRcoM-2) variants with wild-type (WT) BxRcoM-2 in the Fe(III), Fe(II), and Fe(II)CO states
C94S + ferricyanide
C94S + ferricyanide
C94S + ferricyanide
Comparison of electron paramagnetic resonance g values for the CxxS BxRcoM-2 variants and those of WT BxRcoM-2
Characterization of C94S BxRcoM-2
Electronic absorption spectroscopy of purified C94S is strongly suggestive that Cys94 is the Fe(III) heme ligand in wild-type BxRcoM-2. Unlike the C127S or C130S variants, the electronic absorption spectra of C94S are most similar to those of Fe(III) hemes lacking a cysteine(thiolate) ligand (Table 1, Fig. 2, spectra d, e). The Soret band at 418 nm of as-isolated C94S is broader than that of wild-type BxRcoM-2, and the α/β bands at 561 and 532 nm, respectively, are more well defined than those expected for a six-coordinate, low-spin Fe(III) heme with cysteine/histidine axial ligands. The pronounced, characteristic δ band of thiolate ligation is absent; in its place is a weak Soret shoulder near 360 nm. Only one LMCT band is observed, at 640 nm, again suggesting that thiolate ligation is absent. Like the C130S variant, resonance Raman analyses initially indicated that C94S was purified as a mixture of the Fe(II) and Fe(III) oxidation states. In contrast to the C130S variant, however, only a weak EPR signal was initially detected (vide infra). Unlike the C130S variant, oxidation of the C94S variant by potassium ferricyanide resulted in a 3-nm blueshift of the Soret band to 415 nm, a slight decrease in the intensity of the δ band shoulder, and slight shifts in the α/β band positions to 562 and 530 nm, respectively. Additionally, a band at 632 nm appeared, suggesting that a high-spin Fe(III) heme was formed . The observed electronic absorption spectral features are most similar to those of proteins with a mixture of high-spin and low-spin Fe(III) hemes and without a cysteine(thiolate) ligand, such as metmyoglobin, Acetobacter xylinum phosphodiesterase A1 (AxPDEA1), and Methanobacterium thermoautotrophicum direct oxygen sensor (MtDOS) [37, 38]. The electronic absorption spectra of Fe(II) and Fe(II)CO C94S are virtually identical to those of wild-type BxRcoM (Table 1, Figs. S1, S2), suggesting that the reduction and CO binding behaviors of the C94S variant are unperturbed.
The resonance Raman spectrum for as-isolated C94S (Fig. S3, spectrum d) displays oxidation-state and spin-state marker bands consistent with a mixture of oxidation states, similar to those of the C130S variant. Addition of potassium ferricyanide to as-isolated C94S results in a sharpening of the oxidation-state marker band, with one major frequency exhibited at 1,371 cm−1 (Fig. 3, spectrum d), indicative of Fe(III) as the major heme iron oxidation state. A ν3 shoulder, likely due to residual Fe(II), persisted in all of the samples studied. The high-frequency region that contains the spin and coordination-state marker bands (ν3 and ν2) displays broadened bands at 1,493 and 1,503 cm−1 (ν3) and 1,562 and 1,577 cm−1 (ν2). These band positions are indicative of a mixture of low-spin and high-spin Fe(III) hemes, which is most similar to what is observed in the met forms of Fe(III)AxPDEA1 and Fe(III)MtDOS . In addition, the spin-state marker band ν10 is not observed in the resonance Raman spectrum of C94S, unlike the ν10 band of the Fe(III) forms of wild-type, C130S, and C127S BxRcoM-2. Presumably, ν10 in C94S is shifted to lower energy and is hidden owing to overlap with the strong, Gaussian-shaped νc=c band at 1,622 cm−1.
Distinct differences in the low-frequency region of the resonance Raman spectrum of C94S support the conclusion that Cys94 is the native cysteine ligand. In the region where νFe–S is typically seen, there is a band at 304 cm−1 that is sharpened and downshifted relative to the comparable band of wild-type BxRcoM-2 (Fig. 4). This 304-cm−1 mode might plausibly be attributed to an Fe–S(thioether) stretch, since Met104 is the distal Fe(II) ligand; however, we believe that this mode is actually a porphyrin vibration. Gaussian deconvolution of the broad νFe–S bands of Fe(III) wild-type, C130S, and C127S BxRcoM-2 (Fig. 4) generated best fits with three overlapping components. For the C94S spectrum, a single Gaussian was sufficient to fit the sharp band at 304 cm−1. Because the 304-cm−1 component peak is present in the low-energy resonance Raman region of wild-type BxRcoM-2 and all the variants, we assign this band to a porphyrin mode. We attribute the two higher-energy components (313 and 321 cm−1) of Fe(III) wild-type, C130S, and C127S BxRcoM-2 to νFe–S. These νFe–S bands overlap with the porphyrin mode centered at 304 cm−1, like that of cysteine-ligated NPAS2 . A new, intense band at 226 cm−1 is seen in the resonance Raman spectrum of C94S reacted with potassium ferricyanide, which we tentatively assign as the νFe–His stretch of the proximal ligand His74. The rationale for this assignment is the large resonance enhancement of the νFe–His stretch, which typically appears in the 220–270-cm−1 region. This intense feature is typically observed in five- and six-coordinate ferrous hemoproteins with an axial histidine ligand, such as oxymyoglobin, oxyhemoglobin, deoxyhemoglobin, microperoxidase, Bradyrhizobium japonicum FixL, and MtDOS [38, 40, 41, 42]. A similarly intense Fe–His stretch is observed in five-coordinate ferric metmyoglobin .
EPR characterization of C94S provides confirmation that Cys94 is the sixth ligand to the Fe(III) heme in wild-type BxRcoM-2. Data collected for the as-isolated C94S revealed no distinct set of rhombic signals between 2,400 and 3,800 G; however, a low-intensity axial signal at g⊥ = 5.8 was observed, consistent with a high-spin Fe(III) heme. Addition of ferricyanide to the as-isolated form of C94S resulted in an increase in intensity (Fig. 5, spectrum d) of this high-spin axial signal with g⊥ = 5.82 and g|| = 2.00. This axial spectrum is typical of high-spin hemoproteins such as aquo metmyoglobin and high-spin cytochrome c oxidase [44, 45]. The EPR spectrum of the C94S protein (Fig. 5, spectrum d) also contains a small rhombic signal that we attribute to the fraction of low-spin Fe(III). The g anisotropy of this low-spin signal is comparable to that of wild-type BxRcoM, C127S, and C130S, suggesting that Cys127 or Cys130 may replace Cys94 in a fraction of the C94S protein. The fact that the EPR spectrum of C94S exhibits predominantly high-spin Fe(III), and not low-spin Fe(III) like that of wild-type BxRcoM-2, is clear evidence that the cysteine ligand has been lost in this variant. Taken together, our spectral data implicate Cys94 as the distal Fe(III) heme ligand in BxRcoM-2.
Numerous heme proteins are implicated in sensing and transport of small molecules [1, 4, 6]; discrimination among the gaseous signal transducers O2, NO, and CO requires a recognition mechanism that is not based on charge, shape, or size because these gases cannot be readily differentiated by these methods. Instead, heme proteins must modify their affinity for small molecules by altering the iron redox potential, oxidation state, spin state, distal or proximal ligands, or interactions of the gas molecule with residues in the heme-binding pocket . Functional activity of gas-sensing heme proteins is frequently linked to the nature of the heme-binding environment. One element of control utilized to poise the heme for gas binding is a redox-mediated ligand switch; however, the discriminatory binding of gas molecules is modulated by a combination of factors that a redox-mediated ligand switch alone cannot confer. In many cases, a series of events occurs in which the heme iron atom changes oxidation and ligation states preceding gas binding; the final step of regulation is commonly achieved upon stabilization of the correct effector molecule in the heme-binding pocket.
In EcDOS, binding of O2 to heme regulates phosphodiesterase activity [3, 46]. The EcDOS Fe(III) heme, bound within a PAS domain, is coordinated by a histidine residue (His77) and an exogenous water molecule . Reduction of Fe(III) heme to Fe(II) heme results in replacement of the coordinated water by a methionine (Met95) as the distal heme ligand [48, 49]. O2 binding to Fe(II)EcDOS proceeds via replacement of the weakly bound Met95 thioether group. Structural and spectroscopic studies have revealed that charge buildup on the coordinated O2, which does not happen upon CO or NO binding, is neutralized via the guanidinium group of Arg97 [47, 49, 50]. The environment of the heme-binding pocket in EcDOS enables the protein to enhance selectivity for O2 over CO or NO and prevents autoxidation of the heme iron upon O2 binding [3, 50, 51].
HRI, which regulates globin synthesis in reticulocytes in response to available heme levels, is a multidomain heme protein with two distinct heme-binding sites, one of which is a low-spin, six-coordinate heme–thiolate center that functions as an NO sensor [52, 53]. Cys409 of the kinase domain and a histidine (His119 or His120) of the N-terminal domain serve as ligands to this NO-binding heme. Characteristics of cysteine(thiolate) coordination are lost following reduction, suggesting that either a ligand switch or protonation to a cysteine(thiol) occurs to stabilize the more-electron-rich Fe(II) heme [54, 55]. HRI activity is specifically upregulated by formation of a five-coordinate NO adduct, which occurs owing to the strong trans influence of NO and the weakly coordinating nature of the histidine ligand. CO binding opposite the anchoring histidine, forming a six-coordinate species, suppresses activity in HRI, and reaction with O2 results in autoxidation . It is postulated that NO binding releases inhibition by the HRI heme domain, whereas binding of other gas-signaling molecules does not .
RrCooA is the first-known and best-characterized CO gas sensor. This homodimeric protein binds a heme b cofactor within each monomer and utilizes heme-dependent control to regulate expression of genes encoding CO-metabolizing enzymes [20, 57, 58]. The Fe(III) heme in RrCooA is low-spin and six-coordinate; the axial ligands to the Fe(III) heme are a cysteine(thiolate) (Cys75) and the N-terminal proline (Pro2) from the other monomer of the dimer [59, 60, 61]. Reduction of the heme iron results in replacement of the cysteine(thiolate) ligand (Cys75) with histidine (His77) [61, 62]. RrCooA is unusual in that the weakly coordinated N-terminal proline (Pro2) is retained during the redox exchange and thereafter is replaced by the exogenous CO molecule [60, 63]. Binding of CO initiates conformational changes that are propagated along the dimer interface, ultimately resulting in DNA binding [60, 63, 64, 65, 66]. Addition of O2 to Fe(II)RrCooA results in autoxidation, which causes the protein to revert to the Cys–His axial coordination characteristic of the Fe(III) heme . NO reacts with either Fe(III) or Fe(II)RrCooA to form a five-coordinate Fe(II)–NO heme, which is inactive toward DNA binding . Thus, RrCooA is also able to discriminate between O2, CO, and NO, and regulate the biological response of R. rubrum to its environment.
In a manner similar to RrCooA, the isolated PAS-A domain of the mammalian CO sensor NPAS2 bears an Fe(III) heme that is low-spin and is ligated by a histidine/cysteine(thiolate) motif. Upon reduction of the Fe(III) heme, the cysteine(thiolate) ligand (Cys170) is replaced with a strong donor, postulated to be the deprotonated side chain of His171 . One of the histidines is then released to bind CO: whether this is His119 or His171 is not clear. CO-dependent signaling is transduced through the PAS-A domain via disruption of a hydrogen-bonding network, which ultimately causes dissociation of NPAS2 from its heterodimeric partner brain and muscle Arnt-like protein 1 (BMAL1). Dissociation of the DNA-binding NPAS2–BMAL1 complex prevents transcription of per and cry genes, both of which are involved in regulation of the circadian clock [18, 39, 68]. NPAS2 is reported neither to bind O2 reversibly nor to interact with NO . The structural basis for this discrimination is unknown, but it is hypothesized that secondary interactions in the heme-binding pocket contribute.
Recent spectroscopic studies and in vivo functional assays have linked the heme-containing BxRcoM proteins to aerobic CO sensing [21, 25]. Similar to other small-molecule sensor proteins that respond to O2, CO, and NO binding, BxRcoM function is modulated by the oxidation state of the heme iron and by the ligand environment of the heme cofactor. Our prior studies demonstrated that BxRcoM-2 is a cysteine-ligated heme protein that undergoes a redox-mediated ligand switch . In the Fe(III) state, the proximal ligand is inferred to be His74 on the basis of sequence similarity to and site-directed mutagenesis studies of the homologous BxRcoM-1 protein (vide supra) . The BxRcoM-1 and BxRcoM-2 proteins contain three cysteine residues as the potential distal ligand to the Fe(III) heme: Cys94, Cys127, and Cys130. Given the involvement of cysteine(thiolate) residues in redox-mediated ligand switches, which appear to prime gas-regulated hemoproteins for effector-mediated response, we set out to identify the Fe(III)BxRcoM cysteine(thiolate) ligand.
Herein, Cys94 was identified as the distal Fe(III) heme ligand in BxRcoM-2. Sequence alignments of putative RcoM homologues suggested Cys130 was the likelier heme ligand candidate. Three cysteine-to-serine variants were generated, and unlike the other variants studied, the spectral data strongly suggest that C94S does not possess the native low-spin Fe(III) heme with histidine/cysteine(thiolate) coordination. The EPR spectrum of C94S was dominated by an axial signal characteristic of high-spin heme, providing the most compelling evidence that the strongly donating thiolate axial ligand was absent. A minor low-spin component was also observed, suggesting that a sixth ligand may bind in place of Cys94 in a small population of the Fe(III) C94S variant. Given the apparent g anisotropy of this weak low-spin signal, we speculate that Cys130, the most conserved of the three cysteines, or possibly Cys127, may replace the native Cys94. The electronic absorption spectrum of C94S lacked the well-resolved δ band and the two weak LMCT bands in the visible region characteristic of a low-spin thiolate-ligated heme. Furthermore, resonance Raman data suggested that the Fe–S stretch was absent, and a new band at 226 cm−1 was tentatively assigned as a νFe–His mode of a high-spin heme. Thus, all the spectral data point to Cys94 as the heme ligand in BxRcoM-2 although this residue is not highly conserved among RcoM proteins. It remains to be seen whether other RcoM proteins bear heme, whether they employ cysteine coordination, or whether Cys130 plays some other role in signal transduction in these proteins.
The authors thank Thomas C. Brunold (University of Wisconsin–Madison) and his research group for their expertise and the generous use of their instrumentation. Rapid DNA sequencing procedures were performed at the University of Wisconsin–Madison Biotechnology Center facility. Sequence searches utilized both database and analysis functions of the Universal Protein Resource (UniProt) Knowledgebase and Reference Clusters (http://www.uniprot.org) and the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This work was supported by NIH grant GM-53228 to G.P.R. and HL-065217 to J.N.B.
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