Contribution of a surface salt bridge to the protein stability of deep-sea Shewanella benthica cytochrome c'.

Two homologous cytochromes c', SBCP and SVCP, from deep-sea Shewanella benthica and Shewanella violacea respectively exhibit only nine surface amino acid substitutions, along with one at the N-terminus. Despite the small sequence difference, SBCP is thermally more stable than SVCP. Here, we examined the thermal stability of SBCP variants, each containing one of the nine substituted residues in SVCP, and found that the SBCP K87V variant was the most destabilized. We then determined the X-ray crystal structure of the SBCP K87V variant at a resolution of 2.1 Å. The variant retains a four-helix bundle structure similar to the wild-type, but notable differences are observed in the hydration structure around the mutation site. Instead of forming of the intrahelical salt bridge between Lys-87 and Asp-91 in the wild-type, a clathrate-like hydration around Val-87 through a hydrogen bond network with the nearby amino acid residues is observed. This network potentially enhances the ordering of surrounding water molecules, leading to an entropic destabilization of the protein. These results suggest that the unfavorable hydrophobic hydration environment around Val-87 and the inability to form the Asp-91-mediated salt bridge contribute to the observed difference in stability between SBCP and SVCP. These findings will be useful in future protein engineering for controlling protein stability through the manipulation of surface intrahelical salt bridges.

A heme pocket aromatic quadrupole modulates gas binding to cytochrome c'-ß: Implications for NO sensors.

The structural basis by which gas-binding heme proteins control their interactions with NO, CO, and O2 is fundamental to enzymology, biotechnology, and human health. Cytochromes c' (cyts c') are a group of putative NO-binding heme proteins that fall into two families: the well-characterized four alpha helix bundle fold (cyts c'-α) and an unrelated family with a large beta-sheet fold (cyts c'-ß) resembling that of cytochromes P460. A recent structure of cyt c'-ß from Methylococcus capsulatus Bath revealed two heme pocket phenylalanine residues (Phe 32 and Phe 61) positioned near the distal gas-binding site. This feature, dubbed the "Phe cap," is highly conserved within the sequences of other cyts c'-ß but is absent in their close homologs, the hydroxylamine-oxidizing cytochromes P460, although some do contain a single Phe residue. Here, we report an integrated structural, spectroscopic, and kinetic characterization of cyt c'-ß from Methylococcus capsulatus Bath complexes with diatomic gases, focusing on the interaction of the Phe cap with NO and CO. Significantly, crystallographic and resonance Raman data show that orientation of the electron-rich aromatic ring face of Phe 32 toward distally bound NO or CO is associated with weakened backbonding and higher off rates. Moreover, we propose that an aromatic quadrupole also contributes to the unusually weak backbonding reported for some heme-based gas sensors, including the mammalian NO sensor, soluble guanylate cyclase. Collectively, this study sheds light on the influence of highly conserved distal Phe residues on heme-gas complexes of cytochrome c'-ß, including the potential for aromatic quadrupoles to modulate NO and CO binding in other heme proteins.

Crystal structure of thermally stable homodimeric cytochrome c'-ß from Thermus thermophilus.

Cytochrome c'-ß is a heme protein that belongs to the cytochrome P460 family and consists of homodimeric subunits with a predominantly antiparallel ß-sheet fold. Here, the crystal structure of cytochrome c'-ß from the thermophilic Thermus thermophilus (TTCP-ß) is reported at 1.74 Šresolution. TTCP-ß has a typical antiparallel ß-sheet fold similar to that of cytochrome c'-ß from the moderately thermophilic Methylococcus capsulatus (MCCP-ß). The phenylalanine cap structure around the distal side of the heme is also similar in TTCP-ß and MCCP-ß, indicating that both proteins similarly bind nitric oxide and carbon monoxide, as observed spectroscopically. Notably, TTCP-ß exhibits a denaturation temperature of 117°C, which is higher than that of MCCP-ß. Mutational analysis reveals that the increased homodimeric interface area of TTCP-ß contributes to its high thermal stability. Furthermore, 14 proline residues, which are mostly located in the TTCP-ß loop regions, possibly contribute to the rigid loop structure compared with MCCP-ß, which has only six proline residues. These findings, together with those from phylogenetic analysis, suggest that the structures of Thermus cytochromes c'-ß, including TTCP-ß, are optimized for function under the high-temperature conditions in which the source organisms live.

Open-Bundle Structure as the Unfolding Intermediate of Cytochrome c' Revealed by Small Angle Neutron Scattering.

The dynamic structure changes, including the unfolding, dimerization, and transition from the compact to the open-bundle unfolding intermediate structure of Cyt c', were detected by a small-angle neutron scattering experiment (SANS). The structure of Cyt c' was changed into an unstructured random coil at pD = 1.7 (Rg = 25 Å for the Cyt c' monomer). The four-α-helix bundle structure of Cyt c' at neutral pH was transitioned to an open-bundle structure (at pD ~13), which is given by a numerical partial scattering function analysis as a joint-clubs model consisting of four clubs (α-helices) connected by short loops. The compactly folded structure of Cyt c' (radius of gyration, Rg = 18 Å for the Cyt c' dimer) at neutral or mildly alkaline pD transited to a remarkably larger open-bundle structure at pD ~13 (Rg = 25 Å for the Cyt c' monomer). The open-bundle structure was also supported by ab initio modeling.

Thermal stability tuning without affecting gas-binding function of Thermochromatium tepidum cytochrome c'.

Hydrogenophilus thermoluteolus, Thermochromatium tepidum, and Allochromatium vinosum, which grow optimally at 52, 49, and 25 °C, respectively, have homologous cytochromes c' (PHCP, TTCP, and AVCP, respectively) exhibiting at least 50% amino acid sequence identity. Here, the thermal stability of the recombinant TTCP protein was first confirmed to be between those of PHCP and AVCP. Structure comparison of the 3 proteins and a mutagenesis study on TTCP revealed that hydrogen bonds and hydrophobic interactions between the heme and amino acid residues were responsible for their stability differences. In addition, PHCP, TTCP, and AVCP and their variants with altered stability similarly bound nitric oxide and carbon oxide, but not oxygen. Therefore, the thermal stability of TTCP together with PHCP and AVCP can be tuned through specific interactions around the heme without affecting their gas-binding function. These cytochromes c' will be useful as specific gas sensor proteins exhibiting a wide thermal stability range.

Thermal destabilization mechanism of cytochrome c' from psychrophilic Shewanella violacea.

Cytochrome c' is a nitric oxide (NO)-binding heme protein found in Gram negative bacteria. The thermal stability of psychrophilic Shewanella violacea cytochrome c' (SVCP) is lower than those of its homologues from other 2 psychrophilic Shewanella species, indicating that thermal destabilization mechanism for low-temperature adaptation accumulates in SVCP. In order to understand this mechanism at the amino acid level, here the stability and function of SVCP variants, modeled using the 2 homologues, were examined. The variants exhibited increased stability, and they bound NO similar to the wild type. The vulnerability as to the SVCP stability could be attributed to less hydrogen bond at the subunit interface, more flexible loop structure, and less salt bridge on the protein surface, which appear to be its destabilization mechanism. This study provides an example for controlling stability without spoiling function in psychrophilic proteins.

Distinguishing Nitro vs Nitrito Coordination in Cytochrome c' Using Vibrational Spectroscopy and Density Functional Theory.

Nitrite coordination to heme cofactors is a key step in the anaerobic production of the signaling molecule nitric oxide (NO). An ambidentate ligand, nitrite has the potential to coordinate via the N- (nitro) or O- (nitrito) atoms in a manner that can direct its reactivity. Distinguishing nitro vs nitrito coordination, along with the influence of the surrounding protein, is therefore of particular interest. In this study, we probed Fe(III) heme-nitrite coordination in Alcaligenes xylosoxidans cytochrome c' (AXCP), an NO carrier that excludes anions in its native state but that readily binds nitrite (Kd ∼ 0.5 mM) following a distal Leu16 → Gly mutation to remove distal steric constraints. Room-temperature resonance Raman spectra (407 nm excitation) identify ν(Fe-NO2), δ(ONO), and νs(NO2) nitrite ligand vibrations in solution. Illumination with 351 nm UV light results in photoconversion to {FeNO}6 and {FeNO}7 states, enabling FTIR measurements to distinguish νs(NO2) and νas(NO2) vibrations from differential spectra. Density functional theory calculations highlight the connections between heme environment, nitrite coordination mode, and vibrational properties and confirm that nitrite binds to L16G AXCP exclusively through the N atom. Efforts to obtain the nitrite complex crystal structure were hampered by photochemistry in the X-ray beam. Although low dose crystal structures could be modeled with a mixed nitrite (nitro)/H2O distal population, their photosensitivity and partial occupancy underscores the value of the vibrational approach. Overall, this study sheds light on steric determinants of heme-nitrite binding and provides vibrational benchmarks for future studies of heme protein nitrite reactions.

Structural and functional insights into thermally stable cytochrome c' from a thermophile.

Thermophilic Hydrogenophilus thermoluteolus cytochrome c' (PHCP) exhibits higher thermal stability than a mesophilic counterpart, Allochromatium vinosum cytochrome c' (AVCP), which has a homo-dimeric structure and ligand-binding ability. To understand the thermal stability mechanism and ligand-binding ability of the thermally stable PHCP protein, the crystal structure of PHCP was first determined. It formed a homo-dimeric structure, the main chain root mean square deviation (rmsd) value between PHCP and AVCP being 0.65 Å. In the PHCP structure, six specific residues appeared to strengthen the heme-related and subunit-subunit interactions, which were not conserved in the AVCP structure. PHCP variants having altered subunit-subunit interactions were more severely destabilized than ones having altered heme-related interactions. The PHCP structure further revealed a ligand-binding channel and a penta-coordinated heme, as observed in the AVCP protein. A spectroscopic study clearly showed that some ligands were bound to the PHCP protein. It is concluded that the dimeric PHCP from the thermophile is effectively stabilized through heme-related and subunit-subunit interactions with conservation of the ligand-binding ability. BRIEF SUMMARY: We report the X-ray crystal structure of cytochrome c' (PHCP) from thermophilic Hydrogenophilus thermoluteolus. The high thermal stability of PHCP was attributed to heme-related and subunit-subunit interactions, which were confirmed by a mutagenesis study. The ligand-binding ability of PHCP was examined by spectrophotometry. PHCP acquired the thermal stability with conservation of the ligand-binding ability. This study furthers the understanding of the stability and function of cytochromes c.

Formation and carbon monoxide-dependent dissociation of Allochromatium vinosum cytochrome c' oligomers using domain-swapped dimers.

The number of artificial protein supramolecules has been increasing; however, control of protein oligomer formation remains challenging. Cytochrome c' from Allochromatium vinosum (AVCP) is a homodimeric protein in its native form, where its protomer exhibits a four-helix bundle structure containing a covalently bound five-coordinate heme as a gas binding site. AVCP exhibits a unique reversible dimer-monomer transition according to the absence and presence of CO. Herein, domain-swapped dimeric AVCP was constructed and utilized to form a tetramer and high-order oligomers. The X-ray crystal structure of oxidized tetrameric AVCP consisted of two monomer subunits and one domain-swapped dimer subunit, which exchanged the region containing helices αA and αB between protomers. The active site structures of the domain-swapped dimer subunit and monomer subunits in the tetramer were similar to those of the monomer subunits in the native dimer. The subunit-subunit interactions at the interfaces of the domain-swapped dimer and monomer subunits in the tetramer were also similar to the subunit-subunit interaction in the native dimer. Reduced tetrameric AVCP dissociated to a domain-swapped dimer and two monomers upon CO binding. Without monomers, the domain-swapped dimers formed tetramers, hexamers, and higher-order oligomers in the absence of CO, whereas the oligomers dissociated to domain-swapped dimers in the presence of CO, demonstrating that the domain-swapped dimer maintains the CO-induced subunit dissociation behavior of native ACVP. These results suggest that protein oligomer formation may be controlled by utilizing domain swapping for a dimer-monomer transition protein.

Cytochromes c': Structure, Reactivity and Relevance to Haem-Based Gas Sensing.

Cytochromes c' are a group of class IIa cytochromes with pentacoordinate haem centres and are found in photosynthetic, denitrifying and methanotrophic bacteria. Their function remains unclear, although roles in nitric oxide (NO) trafficking during denitrification or in cellular defence against nitrosoative stress have been proposed. Cytochromes c' are typically dimeric with each c-type haem-containing monomer folding as a four-α-helix bundle. Their hydrophobic and crowded distal sites impose severe restrictions on the binding of distal ligands, including diatomic gases. By contrast, NO binds to the proximal haem face in a similar manner to that of the eukaryotic NO sensor, soluble guanylate cyclase and bacterial analogues. In this review, we focus on how structural features of cytochromes c' influence haem spectroscopy and reactivity with NO, CO and O2. We also discuss the relevance of cytochrome c' to understanding the mechanisms of gas binding to haem-based sensor proteins.

Hydrogen bonding of the dissociated histidine ligand is not required for formation of a proximal NO adduct in cytochrome c'.

Cytochromes c', that occur in methanotrophic, denitrifying and photosynthetic bacteria, form unusual proximal penta-coordinate NO complexes via a hexa-coordinate distal NO intermediate. Their NO binding properties are similar to those of the eukaryotic NO sensor, soluble guanylate cyclase, for which they provide a valuable structural model. Previous studies suggested that hydrogen bonding between the displaced proximal histidine (His120) ligand (following its dissociation from heme due to trans effects from the distally bound NO) and a conserved aspartate residue (Asp121) could play a key role in allowing proximal NO binding to occur. We have characterized three variants of Alcaligenes xylosoxidans cytochrome c' (AXCP) where Asp121 has been replaced by Ala, Ile and Gln, respectively. In all variants, hydrogen bonding between residue 121 and His120 is abolished yet 5-coordinate proximal NO species are still formed. Our data therefore demonstrate that the His120-Asp121 bond is not essential for proximal NO binding although it likely provides an energy minimum for the displaced His ligand. All variants have altered proximal pocket structure relative to native AXCP.

Resonance Raman Spectra of Five-Coordinate Heme-Nitrosyl Cytochromes c': Effect of the Proximal Heme-NO Environment.

Five-coordinate heme nitrosyl complexes (5cNO) underpin biological heme-NO signal transduction. Bacterial cytochromes c' are some of the few structurally characterized 5cNO proteins, exhibiting a distal to proximal 5cNO transition of relevance to NO sensing. Establishing how 5cNO coordination (distal vs proximal) depends on the heme environment is important for understanding this process. Recent 5cNO crystal structures of Alcaligenes xylosoxidans cytochrome c' (AXCP) and Shewanella frigidimarina cytochrome c' (SFCP) show a basic residue (Arg124 and Lys126, respectively) near the proximal NO binding sites. Using resonance Raman (RR) spectroscopy, we show that structurally characterized 5cNO complexes of AXCP variants and SFCP exhibit a range of ν(NO) (1651-1671 cm(-1)) and ν(FeNO) (519-536 cm(-1)) vibrational frequencies, depending on the nature of the proximal heme pocket and the sample temperature. While the AXCP Arg124 residue appears to have little impact on 5cNO vibrations, the ν(NO) and ν(FeNO) frequencies of the R124K variant are consistent with (electrostatically) enhanced Fe(II) → (NO)π* backbonding. Notably, RR frequencies for SFCP and R124A AXCP are significantly displaced from the backbonding trendline, which in light of recent crystallographic data and density functional theory modeling may reflect changes in the Fe-N-O angle and/or extent of σ-donation from the NO(π*) to the Fe(II) (dz(2)) orbital. For R124A AXCP, correlation of vibrational and crystallographic data is complicated by distal and proximal 5cNO populations. Overall, this study highlights the complex structure-vibrational relationships of 5cNO proteins that allow RR spectra to distinguish 5cNO coordination in certain electrostatic and steric environments.

Conformational control of the binding of diatomic gases to cytochrome c'.

The cytochromes c' (CYTcp) are found in denitrifying, methanotrophic and photosynthetic bacteria. These proteins are able to form stable adducts with CO and NO but not with O2. The binding of NO to CYTcp currently provides the best structural model for the NO activation mechanism of soluble guanylate cyclase. Ligand binding in CYTcps has been shown to be highly dependent on residues in both the proximal and distal heme pockets. Group 1 CYTcps typically have a phenylalanine residue positioned close to the distal face of heme, while for group 2, this residue is typically leucine. We have structurally, spectroscopically and kinetically characterised the CYTcp from Shewanella frigidimarina (SFCP), a protein that has a distal phenylalanine residue and a lysine in the proximal pocket in place of the more common arginine. Each monomer of the SFCP dimer folds as a 4-alpha-helical bundle in a similar manner to CYTcps previously characterised. SFCP exhibits biphasic binding kinetics for both NO and CO as a result of the high level of steric hindrance from the aromatic side chain of residue Phe 16. The binding of distal ligands is thus controlled by the conformation of the phenylalanine ring. Only a proximal 5-coordinate NO adduct, confirmed by structural data, is observed with no detectable hexacoordinate distal NO adduct.

The roles of C-terminal residues on the thermal stability and local heme environment of cytochrome c' from the thermophilic purple sulfur bacterium Thermochromatium tepidum.

A soluble cytochrome (Cyt) c' from thermophilic purple sulfur photosynthetic bacterium Thermochromatium (Tch.) tepidum exhibits marked thermal tolerance compared with that from the closely related mesophilic counterpart Allochromatium vinosum. Here, we focused on the difference in the C-terminal region of the two Cyts c' and examined the effects of D131 and R129 mutations on the thermal stability and local heme environment of Cyt c' by differential scanning calorimetry (DSC) and resonance Raman (RR) spectroscopy. In the oxidized forms, D131K and D131G mutants exhibited denaturing temperatures significantly lower than that of the recombinant control Cyt c'. In contrast, R129K and R129A mutants denatured at nearly identical temperatures with the control Cyt c', indicating that the C-terminal D131 is an important residue maintaining the enhanced thermal stability of Tch. tepidum Cyt c'. The control Cyt c' and all of the mutants increased their thermal stability upon the reduction. Interestingly, D131K exhibited narrow DSC curves and unusual thermodynamic parameters in both redox states. The RR spectra of the control Cyt c' exhibited characteristic bands at 1,635 and 1,625 cm(-1), ascribed to intermediate spin (IS) and high spin (HS) states, respectively. The IS/HS distribution was differently affected by the D131 and R129 mutations and pH changes. Furthermore, R129 mutants suggested the lowering of their redox potentials. These results strongly indicate that the D131 and R129 residues play significant roles in maintaining the thermal stability and modulating the local heme environment of Tch. tepidum Cyt c'.

Gates and binding pockets for nitric oxide with cytochrome c', according to molecular dynamics.

Random-acceleration molecular-dynamics (RAMD) simulations with models of homodimeric 6-ligated distal-NO and 5-ligated proximal-NO cytochrome c' complexes, in TIP3 H2 O, showed two distinct, non-intercommunicating worlds. In the framework of a long cavity formed by four protein helices with heme at one extremity, NO was observed to follow different pathways with the two complexes to reach the solvent. With the 6-ligated complex, NO was observed to progress by exploiting protein internal channels created by thermal fluctuations, and be temporarily trapped into binding pockets before reaching the preferred gate at the heme end of the cavity. In contrast, with the 5-ligated complex, NO was observed to surface the solvent-exposed helix 7, up to a gate at the other extremity of the protein, only occasionally finding an earlier, direct way out toward the solvent. That only bulk NO gets involved in forming the 5-ligated proximal-NO complex is in agreement with previous experimental observations, while the occurrence of binding pockets suggests that also reservoir NO might play a role with the distal-NO complex.

High thermal stability and unique trimer formation of cytochrome c' from thermophilic Hydrogenophilus thermoluteolus.

Sequence analysis indicated that thermophilic Hydrogenophilus thermoluteolus cytochrome c' (PHCP) and its mesophilic homolog, Allochromatium vinosum cytochrome c' (AVCP), closely resemble each other in a phylogenetic tree of the cytochrome c' family, with 55% sequence identity. The denaturation temperature of PHCP was 87 °C, 35 °C higher than that of AVCP. Furthermore, PHCP exhibited a larger enthalpy change value during its thermal denaturation than AVCP. While AVCP was dimeric, as observed previously, PHCP was trimeric, and this was the first observation as a cytochrome c'. Dissociation of trimeric PHCP and its protein denaturation reversibly occurred at the same time in a two-state transition manner. Therefore, PHCP is enthalpically more stable than AVCP, perhaps due to its unique trimeric form, in addition to the lower number of Gly residues in its putative α-helical regions.

Structure analysis and comparative characterization of the cytochrome c' and flavocytochrome c from thermophilic purple photosynthetic bacterium Thermochromatium tepidum.

The thermodynamic and spectroscopic properties of two soluble electron transport proteins, cytochrome (Cyt) c' and flavocytochrome c, isolated from thermophilic purple sulfur bacterium Thermochromatium (Tch.) tepidum were examined and compared with those of the corresponding proteins from a closely related mesophilic bacterium Allochromatium (Alc.) vinosum. These proteins share sequence identities of 82% for the cytochromes c' and 86% for the flavocytochromes c. Crystal structures of the two proteins have been determined at high resolutions. Differential scanning calorimetry and denaturing experiments show that both proteins from Tch. tepidum are thermally and structurally much more stable than their mesophilic counterparts. The denaturation temperature of Tch. tepidum Cyt c' was 22 °C higher than that of Alc. vinosum Cyt c', and the midpoints of denaturation using guanidine hydrochloride were 2.0 and 1.2 M for the Tch. tepidum and Alc. vinosum flavocytochromes c, respectively. The enhanced stabilities can be interpreted on the basis of the structural and sequence information obtained in this study: increased number of hydrogen bonds formed between main chain nitrogen and oxygen atoms, more compact structures and reduced number of glycine residues. Many residues with large side chains in Alc. vinosum Cyt c' are substituted by alanines in Tch. tepidum Cyt c'. Both proteins from Tch. tepidum exhibit high structural similarities to their counterparts from Alc. vinosum, and the different residues between the corresponding proteins are mainly located on the surface and exposed to the solvent. Water molecules are found in the heme vicinity of Tch. tepidum Cyt c' and form hydrogen bonds with the heme ligand and C-terminal charged residues. Similar bound waters are also found in the vicinity of one heme group in the diheme subunit of Tch. tepidum flavocytochrome c. Electron density map of the Tch. tepidum flavocytochrome c clearly revealed the presence of disulfur atoms positioned between two cysteine residues at the active site near the FAD prosthetic group. The result strongly suggests that flavocytochrome c is involved in the sulfide oxidation in vivo. Detailed discussion is given on the relationships between the crystal structures and the spectroscopic properties observed for these proteins.

How do heme-protein sensors exclude oxygen? Lessons learned from cytochrome c', Nostoc puntiforme heme nitric oxide/oxygen-binding domain, and soluble guanylyl cyclase.

SIGNIFICANCE: Ligand selectivity for dioxygen (O(2)), carbon monoxide (CO), and nitric oxide (NO) is critical for signal transduction and is tailored specifically for each heme-protein sensor. Key NO sensors, such as soluble guanylyl cyclase (sGC), specifically recognized low levels of NO and achieve a total O(2) exclusion. Several mechanisms have been proposed to explain the O(2) insensitivity, including lack of a hydrogen bond donor and negative electrostatic fields to selectively destabilize bound O(2), distal steric hindrance of all bound ligands to the heme iron, and restriction of in-plane movements of the iron atom. RECENT ADVANCES: Crystallographic structures of the gas sensors, Thermoanaerobacter tengcongensis heme-nitric oxide/oxygen-binding domain (Tt H-NOX(1)) or Nostoc puntiforme (Ns) H-NOX, and measurements of O(2) binding to site-specific mutants of Tt H-NOX and the truncated ß subunit of sGC suggest the need for a H-bonding donor to facilitate O(2) binding. CRITICAL ISSUES: However, the O(2) insensitivity of full length sGC with a site-specific replacement of isoleucine by a tyrosine on residue 145 and the very slow autooxidation of Ns H-NOX and cytochrome c' suggest that more complex mechanisms have evolved to exclude O(2) but retain high affinity NO binding. A combined graphical analysis of ligand binding data for libraries of heme sensors, globins, and model heme shows that the NO sensors dramatically inhibit the formation of six-coordinated NO, CO, and O(2) complexes by direct distal steric hindrance (cyt c'), proximal constraints of in-plane iron movement (sGC), or combinations of both following a sliding scale rule. High affinity NO binding in H-NOX proteins is achieved by multiple NO binding steps that produce a high affinity five-coordinate NO complex, a mechanism that also prevents NO dioxygenation. FUTURE DIRECTIONS: Knowledge advanced by further extensive test of this "sliding scale rule" hypothesis should be valuable in guiding novel designs for heme based sensors.

Carbon monoxide poisoning is prevented by the energy costs of conformational changes in gas-binding haemproteins.

Carbon monoxide (CO) is a product of haem metabolism and organisms must evolve strategies to prevent endogenous CO poisoning of haemoproteins. We show that energy costs associated with conformational changes play a key role in preventing irreversible CO binding. AxCYTcp is a member of a family of haem proteins that form stable 5c-NO and 6c-CO complexes but do not form O(2) complexes. Structure of the AxCYTcp-CO complex at 1.25 Å resolution shows that CO binds in two conformations moderated by the extent of displacement of the distal residue Leu16 toward the haem 7-propionate. The presence of two CO conformations is confirmed by cryogenic resonance Raman data. The preferred linear Fe-C-O arrangement (170 ± 8°) is accompanied by a flip of the propionate from the distal to proximal face of the haem. In the second conformation, the Fe-C-O unit is bent (158 ± 8°) with no flip of propionate. The energetic cost of the CO-induced Leu-propionate movements is reflected in a 600 mV (57.9 kJ mol(-1)) decrease in haem potential, a value in good agreement with density functional theory calculations. Substitution of Leu by Ala or Gly (structures determined at 1.03 and 1.04 Å resolutions) resulted in a haem site that binds CO in the linear mode only and where no significant change in redox potential is observed. Remarkably, these variants were isolated as ferrous 6c-CO complexes, attributable to the observed eight orders of magnitude increase in affinity for CO, including an approximately 10,000-fold decrease in the rate of dissociation. These new findings have wide implications for preventing CO poisoning of gas-binding haem proteins.

Heterologous synthesis of cytochrome c' by Escherichia coli is not dependent on the System I cytochrome c biogenesis machinery.

Hydrogenophilus thermoluteolus cytochrome c' (PHCP) has typical spectral properties previously observed for other cytochromes c', which comprise Ambler's class II cytochromes c. The PHCP protein sequence (135 amino acids) deduced from the cloned gene is the most homologous (55% identity) to that of cytochrome c' from Allochromatium vinosum (AVCP). These findings indicate that PHCP forms a four-helix bundle structure, similar to AVCP. Strikingly, PHCP with a covalently bound heme was heterologously synthesized in the periplasm of Escherichia coli strains deficient in the DsbD protein, a component of the System I cytochrome c biogenesis machinery. The heterologous synthesis of PHCP by aerobically growing E. coli also occurred without a plasmid carrying the genes for Ccm proteins, other components of the System I machinery. Unlike Ambler's class I general cytochromes c, the synthesis of PHCP is not dependent on the System I machinery and exhibits similarity to that of E. coli periplasmic cytochrome b(562), a 106-residue four-helix bundle.