Elucidating Electron Storage and Distribution within the Pentaheme Scaffold of Cytochrome c Nitrite Reductase (NrfA).

Cytochrome c nitrite reductases (CcNIR or NrfA) play important roles in the global nitrogen cycle by conserving the usable nitrogen in the soil. Here, the electron storage and distribution properties within the pentaheme scaffold of Geobacter lovleyi NrfA were investigated via electron paramagnetic resonance (EPR) spectroscopy coupled with chemical titration experiments. Initially, a chemical reduction method was established to sequentially add electrons to the fully oxidized protein, 1 equiv at a time. The step-by-step reduction of the hemes was then followed using ultraviolet-visible absorption and EPR spectroscopy. EPR spectral simulations were used to elucidate the sequence of heme reduction within the pentaheme scaffold of NrfA and identify the signals of all five hemes in the EPR spectra. Electrochemical experiments ascertain the reduction potentials for each heme, observed in a narrow range from +10 mV (heme 5) to -226 mV (heme 3) (vs the standard hydrogen electrode). On the basis of quantitative analysis and simulation of the EPR data, we demonstrate that hemes 4 and 5 are reduced first (before the active site heme 1) and serve the purpose of an electron storage unit within the protein. To probe the role of the central heme 3, an H108M NrfA variant was generated where the reduction potential of heme 3 is shifted positively (from -226 to +48 mV). The H108M mutation significantly impacts the distribution of electrons within the pentaheme scaffold and the reduction potentials of the hemes, reducing the catalytic activity of the enzyme to 1% compared to that of the wild type. We propose that this is due to heme 3's important role as an electron gateway in the wild-type enzyme.

Cytochrome c nitrite reductase from the bacterium Geobacter lovleyi represents a new NrfA subclass.

Cytochrome c nitrite reductase (NrfA) catalyzes the reduction of nitrite to ammonium in the dissimilatory nitrate reduction to ammonium (DNRA) pathway, a process that competes with denitrification, conserves nitrogen, and minimizes nutrient loss in soils. The environmental bacterium Geobacter lovleyi has recently been recognized as a key driver of DNRA in nature, but its enzymatic pathway is still uncharacterized. To address this limitation, here we overexpressed, purified, and characterized G. lovleyi NrfA. We observed that the enzyme crystallizes as a dimer but remains monomeric in solution. Importantly, its crystal structure at 2.55-Å resolution revealed the presence of an arginine residue in the region otherwise occupied by calcium in canonical NrfA enzymes. The presence of EDTA did not affect the activity of G. lovleyi NrfA, and site-directed mutagenesis of this arginine reduced enzymatic activity to <3% of the WT levels. Phylogenetic analysis revealed four separate emergences of Arg-containing NrfA enzymes. Thus, the Ca2+-independent, Arg-containing NrfA from G. lovleyi represents a new subclass of cytochrome c nitrite reductase. Most genera from the exclusive clades of Arg-containing NrfA proteins are also represented in clades containing Ca2+-dependent enzymes, suggesting convergent evolution.

Trapping of a Putative Intermediate in the Cytochrome c Nitrite Reductase (ccNiR)-Catalyzed Reduction of Nitrite: Implications for the ccNiR Reaction Mechanism.

Cytochrome c nitrite reductase (ccNiR) is a periplasmic, decaheme homodimeric enzyme that catalyzes the six-electron reduction of nitrite to ammonia. Under standard assay conditions catalysis proceeds without detected intermediates, and it has been assumed that this is also true in vivo. However, this report demonstrates that it is possible to trap a putative intermediate by controlling the electrochemical potential at which reduction takes place. UV/vis spectropotentiometry showed that nitrite-loaded Shewanella oneidensis ccNiR is reduced in a concerted two-electron step to generate an {FeNO}7 moiety at the active site, with an associated midpoint potential of +246 mV vs SHE at pH 7. By contrast, cyanide-bound active site reduction is a one-electron process with a midpoint potential of +20 mV, and without a strong-field ligand the active site midpoint potential shifts 70 mV lower still. EPR analysis subsequently revealed that the {FeNO}7 moiety possesses an unusual spectral signature, different from those normally observed for {FeNO}7 hemes, that may indicate magnetic interaction of the active site with nearby hemes. Protein film voltammetry experiments previously showed that catalytic nitrite reduction to ammonia by S. oneidensis ccNiR requires an applied potential of at least -120 mV, well below the midpoint potential for {FeNO}7 formation. Thus, it appears that an {FeNO}7 active site is a catalytic intermediate in the ccNiR-mediated reduction of nitrite to ammonia, whose degree of accumulation depends exclusively on the applied potential. At low potentials the species is rapidly reduced and does not accumulate, while at higher potentials it is trapped, thus preventing catalytic ammonia formation.

Epsilonproteobacterial hydroxylamine oxidoreductase (εHao): characterization of a 'missing link' in the multihaem cytochrome c family.

Members of the multihaem cytochrome c family such as pentahaem cytochrome c nitrite reductase (NrfA) or octahaem hydroxylamine oxidoreductase (Hao) are involved in various microbial respiratory electron transport chains. Some members of the Hao subfamily, here called εHao proteins, have been predicted from the genomes of nitrate/nitrite-ammonifying bacteria that usually lack NrfA. Here, εHao proteins from the host-associated Epsilonproteobacteria Campylobacter fetus and Campylobacter curvus and the deep-sea hydrothermal vent bacteria Caminibacter mediatlanticus and Nautilia profundicola were purified as εHao-maltose binding protein fusions produced in Wolinella succinogenes. All four proteins were able to catalyze reduction of nitrite (yielding ammonium) and hydroxylamine whereas hydroxylamine oxidation was negligible. The introduction of a tyrosine residue at a position known to cause covalent trimerization of Hao proteins did neither stimulate hydroxylamine oxidation nor generate the Hao-typical absorbance maximum at 460 nm. In most cases, the εHao-encoding gene haoA was situated downstream of haoC, which predicts a tetrahaem cytochrome c of the NapC/NrfH family. This suggested the formation of a membrane-bound HaoCA assembly reminiscent of the menaquinol-oxidizing NrfHA complex. The results indicate that εHao proteins form a subfamily of ammonifying cytochrome c nitrite reductases that represents a 'missing link' in the evolution of NrfA and Hao enzymes.

In meso crystal structure of a novel membrane-associated octaheme cytochrome c from the Crenarchaeon Ignicoccus hospitalis.

The Crenarchaeon Ignicoccus hospitalis lives in symbiosis with Nanoarchaeum equitans providing essential cell components and nutrients to its symbiont. Ignicoccus hospitalis shows an intriguing morphology that points toward an evolutionary role in driving compartmentalization. Therefore, the bioenergetics of this archaeal host-symbiont system remains a pressing question. To date, the only electron acceptor described for I. hospitalis is elemental sulfur, but the organism comprises genes that encode for enzymes involved in nitrogen metabolism, e.g., one nitrate reductase and two octaheme cytochrome c, Igni_0955 (IhOCC) and Igni_1359. Herein, we detail functional and structural studies of the highly abundant IhOCC, including an X-ray crystal structure at 1.7 Å resolution, the first three-dimensional structure of an archaeal OCC. The trimeric IhOCC is membrane associated and exhibits significant structural and functional differences to previously characterized homologs within the hydroxylamine oxidoreductases (HAOs) and octaheme cytochrome c nitrite reductases (ONRs). The positions and spatial arrangement of the eight hemes are highly conserved, but the axial ligands of the individual hemes 3, 6 and 7 and the protein environment of the active site show significant differences. Most notably, the active site heme 4 lacks porphyrin-tyrosine cross-links present in the HAO family. We show that IhOCC efficiently reduces nitrite and hydroxylamine, with possible relevance to detoxification or energy conservation. DATABASE: Structural data are available in the Protein Data Bank under the accession number 4QO5.

Six-electron reduction of nitrite to ammonia by cytochrome c nitrite reductase: insights from density functional theory studies.

In this Forum Article, an extensive discussion of the mechanism of six-electron, seven-proton nitrite reduction by the cytochrome c nitrite reductase enzyme is presented. On the basis of previous studies, the entire mechanism is summarized and a unified picture of the most plausible sequence of elementary steps is presented. According to this scheme, the mechanism can be divided into five functional stages. The first phase of the reaction consists of substrate binding and N-O bond cleavage. Here His277 plays a crucial role as a proton donor. In this step, the N-O bond is cleaved heterolytically through double protonation of the substrate. The second phase of the mechanism consists of two proton-coupled electron-transfer events, leading to an HNO intermediate. The third phase involves the formation of hydroxylamine, where Arg114 provides the necessary proton for the reaction. The second N-O bond is cleaved in the fourth phase of the mechanism, again triggered by proton transfer from His277. The Tyr218 side chain governs the fifth and last phase of the mechanism. It consists of radical transfer and ammonia formation. Thus, this mechanism implies that all conserved active-site side chains work in a concerted way in order to achieve this complex chemical transformation from nitrite to ammonia. The Forum Article also provides a detailed discussion of the density functional theory based cluster model approach to bioinorganic reactivity. A variety of questions are considered: the resting state of enzyme and substrate binding modes, interaction with the metal site and with active-site side chains, electron- and proton-transfer events, substrate dissociation, etc.

Regulation of Nitrite Stress Response in Desulfovibrio vulgaris Hildenborough, a Model Sulfate-Reducing Bacterium.

UNLABELLED: Sulfate-reducing bacteria (SRB) are sensitive to low concentrations of nitrite, and nitrite has been used to control SRB-related biofouling in oil fields. Desulfovibrio vulgaris Hildenborough, a model SRB, carries a cytochrome c-type nitrite reductase (nrfHA) that confers resistance to low concentrations of nitrite. The regulation of this nitrite reductase has not been directly examined to date. In this study, we show that DVU0621 (NrfR), a sigma54-dependent two-component system response regulator, is the positive regulator for this operon. NrfR activates the expression of the nrfHA operon in response to nitrite stress. We also show that nrfR is needed for fitness at low cell densities in the presence of nitrite because inactivation of nrfR affects the rate of nitrite reduction. We also predict and validate the binding sites for NrfR upstream of the nrfHA operon using purified NrfR in gel shift assays. We discuss possible roles for NrfR in regulating nitrate reductase genes in nitrate-utilizing Desulfovibrio spp. IMPORTANCE: The NrfA nitrite reductase is prevalent across several bacterial phyla and required for dissimilatory nitrite reduction. However, regulation of the nrfA gene has been studied in only a few nitrate-utilizing bacteria. Here, we show that in D. vulgaris, a bacterium that does not respire nitrate, the expression of nrfHA is induced by NrfR upon nitrite stress. This is the first report of regulation of nrfA by a sigma54-dependent two-component system. Our study increases our knowledge of nitrite stress responses and possibly of the regulation of nitrate reduction in SRB.

An ethane-bridged porphyrin dimer as a model of di-heme proteins: inorganic and bioinorganic perspectives and consequences of heme-heme interactions.

Interaction between heme centers has been cleverly implemented by Nature in order to regulate different properties of multiheme cytochromes, thereby allowing them to perform a wide variety of functions. Our broad interest lies in unmasking the roles played by heme-heme interactions in modulating different properties viz., metal spin state, redox potential etc., of the individual heme centers using an ethane-bridged porphyrin dimer as a synthetic model of dihemes. The large differences in the structure and properties of the diheme complexes, as compared to the monoheme analogs, provide unequivocal evidence of the role played by heme-heme interactions in the dihemes. This Perspective provides a brief account of our recent efforts to explore these interesting aspects and the subsequent outcomes.

Correlations between the Electronic Properties of Shewanella oneidensis Cytochrome c Nitrite Reductase (ccNiR) and Its Structure: Effects of Heme Oxidation State and Active Site Ligation.

The electrochemical properties of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR), a homodimer that contains five hemes per protomer, were investigated by UV-visible and electron paramagnetic resonance (EPR) spectropotentiometries. Global analysis of the UV-vis spectropotentiometric results yielded highly reproducible values for the heme midpoint potentials. These midpoint potential values were then assigned to specific hemes in each protomer (as defined in previous X-ray diffraction studies) by comparing the EPR and UV-vis spectropotentiometric results, taking advantage of the high sensitivity of EPR spectra to the structural microenvironment of paramagnetic centers. Addition of the strong-field ligand cyanide led to a 70 mV positive shift of the active site's midpoint potential, as the cyanide bound to the initially five-coordinate high-spin heme and triggered a high-spin to low-spin transition. With cyanide present, three of the remaining hemes gave rise to distinctive and readily assignable EPR spectral changes upon reduction, while a fourth was EPR-silent. At high applied potentials, interpretation of the EPR spectra in the absence of cyanide was complicated by a magnetic interaction that appears to involve three of five hemes in each protomer. At lower applied potentials, the spectra recorded in the presence and absence of cyanide were similar, which aided global assignment of the signals. The midpoint potential of the EPR-silent heme could be assigned by default, but the assignment was also confirmed by UV-vis spectropotentiometric analysis of the H268M mutant of ccNiR, in which one of the EPR-silent heme's histidine axial ligands was replaced with a methionine.

Structural study of the X-ray-induced enzymatic reaction of octahaem cytochrome C nitrite reductase.

Octahaem cytochrome c nitrite reductase from the bacterium Thioalkalivibrio nitratireducens catalyzes the reduction of nitrite to ammonium and of sulfite to sulfide. The reducing properties of X-ray radiation and the high quality of the enzyme crystals allow study of the catalytic reaction of cytochrome c nitrite reductase directly in a crystal of the enzyme, with the reaction being induced by X-rays. Series of diffraction data sets with increasing absorbed dose were collected from crystals of the free form of the enzyme and its complexes with nitrite and sulfite. The corresponding structures revealed gradual changes associated with the reduction of the catalytic haems by X-rays. In the case of the nitrite complex the conversion of the nitrite ions bound in the active sites to NO species was observed, which is the beginning of the catalytic reaction. For the free form, an increase in the distance between the oxygen ligand bound to the catalytic haem and the iron ion of the haem took place. In the case of the sulfite complex no enzymatic reaction was detected, but there were changes in the arrangement of the active-site water molecules that were presumably associated with a change in the protonation state of the sulfite ions.

Resolution of key roles for the distal pocket histidine in cytochrome C nitrite reductases.

Cytochrome c nitrite reductases perform a key step in the biogeochemical N-cycle by catalyzing the six-electron reduction of nitrite to ammonium. These multiheme cytochromes contain a number of His/His ligated c-hemes for electron transfer and a structurally differentiated heme that provides the catalytic center. The catalytic heme has proximal ligation from lysine, or histidine, and an exchangeable distal ligand bound within a pocket that includes a conserved histidine. Here we describe properties of a penta-heme cytochrome c nitrite reductase in which the distal His has been substituted by Asn. The variant is unable to catalyze nitrite reduction despite retaining the ability to reduce a proposed intermediate in that process, namely, hydroxylamine. A combination of electrochemical, structural and spectroscopic studies reveals that the variant enzyme simultaneously binds nitrite and electrons at the catalytic heme. As a consequence the distal His is proposed to play a key role in orienting the nitrite for N-O bond cleavage. The electrochemical experiments also reveal that the distal His facilitates rapid nitrite binding to the catalytic heme of the native enzyme. Finally it is noted that the thermodynamic descriptions of nitrite- and electron-binding to the active site of the variant enzyme are modulated by the prevailing oxidation states of the His/His ligated hemes. This behavior is likely to be displayed by other multicentered redox enzymes such that there are wide implications for considering the determinants of catalytic activity in this important and varied group of oxidoreductases.

Hydrogen bonding networks tune proton-coupled redox steps during the enzymatic six-electron conversion of nitrite to ammonia.

Multielectron multiproton reactions play an important role in both biological systems and chemical reactions involved in energy storage and manipulation. A key strategy employed by nature in achieving such complex chemistry is the use of proton-coupled redox steps. Cytochrome c nitrite reductase (ccNiR) catalyzes the six-electron seven-proton reduction of nitrite to ammonia. While a catalytic mechanism for ccNiR has been proposed on the basis of studies combining computation and crystallography, there have been few studies directly addressing the nature of the proton-coupled events that are predicted to occur along the nitrite reduction pathway. Here we use protein film voltammetry to directly interrogate the proton-coupled steps that occur during nitrite reduction by ccNiR. We find that conversion of nitrite to ammonia by ccNiR adsorbed to graphite electrodes is defined by two distinct phases; one is proton-coupled, and the other is not. Mutation of key active site residues (H257, R103, and Y206) modulates these phases and specifically alters the properties of the detected proton-dependent step but does not inhibit the ability of ccNiR to conduct the full reduction of nitrite to ammonia. We conclude that the active site residues examined are responsible for tuning the protonation steps that occur during catalysis, likely through an extensive hydrogen bonding network, but are not necessarily required for the reaction to proceed. These results provide important insight into how enzymes can specifically tune proton- and electron transfer steps to achieve high turnover numbers in a physiological pH range.

Contribution of the NO-detoxifying enzymes HmpA, NorV and NrfA to nitrosative stress protection of Salmonella Typhimurium in raw sausages.

The antimicrobial action of the curing agent sodium nitrite (NaNO2) in raw sausage fermentation is thought to mainly depend on the release of cytotoxic nitric oxide (NO) at acidic pH. Salmonella Typhimurium is capable of detoxifying NO via the flavohemoglobin HmpA, the flavorubredoxin NorV and the periplasmic cytochrome C nitrite reductase NrfA. In this study, the contribution of these systems to nitrosative stress tolerance in raw sausages was investigated. In vitro growth assays of the S. Typhimurium 14028 deletion mutants ΔhmpA, ΔnorV and ΔnrfA revealed a growth defect of ΔhmpA in the presence of acidified NaNO2. Transcriptional analysis of the genes hmpA, norV and nrfA in the wild-type showed a 41-fold increase in hmpA transcript levels in the presence of 150 mg/l acidified NaNO2, whereas transcription of norV and nrfA was not enhanced. However, challenge assays performed with short-ripened spreadable sausages produced with 0 or 150 mg/kg NaNO2 failed to reveal a phenotype for any of the mutants compared to the wild-type. Hence, none of the NO detoxification systems HmpA, NorV and NrfA is solely responsible for nitrosative stress tolerance of S. Typhimurium in raw sausages. Whether these systems act cooperatively, or if there are other yet undescribed mechanisms involved is currently unknown.

Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) does not disproportionate hydroxylamine to ammonia and nitrite, despite a strongly favorable driving force.

Cytochrome c nitrite reductase (ccNiR) from Shewanella oneidensis, which catalyzes the six-electron reduction of nitrite to ammonia in vivo, was shown to oxidize hydroxylamine in the presence of large quantities of this substrate, yielding nitrite as the sole free nitrogenous product. UV-visible stopped-flow and rapid-freeze-quench electron paramagnetic resonance data, along with product analysis, showed that the equilibrium between hydroxylamine and nitrite is fairly rapidly established in the presence of high initial concentrations of hydroxylamine, despite said equilibrium lying far to the left. By contrast, reduction of hydroxylamine to ammonia did not occur, even though disproportionation of hydroxylamine to yield both nitrite and ammonia is strongly thermodynamically favored. This suggests a kinetic barrier to the ccNiR-catalyzed reduction of hydroxylamine to ammonia. A mechanism for hydroxylamine reduction is proposed in which the hydroxide group is first protonated and released as water, leaving what is formally an NH2(+) moiety bound at the heme active site. This species could be a metastable intermediate or a transition state but in either case would exist only if it were stabilized by the donation of electrons from the ccNiR heme pool into the empty nitrogen p orbital. In this scenario, ccNiR does not catalyze disproportionation because the electron-donating hydroxylamine does not poise the enzyme at a sufficiently low potential to stabilize the putative dehydrated hydroxylamine; presumably, a stronger reductant is required for this.

Heme-bound nitroxyl, hydroxylamine, and ammonia ligands as intermediates in the reaction cycle of cytochrome c nitrite reductase: a theoretical study.

In this article, we consider, in detail, the second half-cycle of the six-electron nitrite reduction mechanism catalyzed by cytochrome c nitrite reductase. In total, three electrons and four protons must be provided to reach the final product, ammonia, starting from the HNO intermediate. According to our results, the first event in this half-cycle is the reduction of the HNO intermediate, which is accomplished by two PCET reactions. Two isomeric radical intermediates, HNOH(•) and H2NO(•), are formed. Both intermediates are readily transformed into hydroxylamine, most likely through intramolecular proton transfer from either Arg114 or His277. An extra proton must enter the active site of the enzyme to initiate heterolytic cleavage of the N-O bond. As a result of N-O bond cleavage, the H2N(+) intermediate is formed. The latter readily picks up an electron, forming H2N(+•), which in turn reacts with Tyr218. Interestingly, evidence for Tyr218 activity was provided by the mutational studies of Lukat (Biochemistry 47:2080, 2008), but this has never been observed in the initial stages of the overall reduction process. According to our results, an intramolecular reaction with Tyr218 in the final step of the nitrite reduction process leads directly to the final product, ammonia. Dissociation of the final product proceeds concomitantly with a change in spin state, which was also observed in the resonance Raman investigations of Martins et al. (J Phys Chem B 114:5563, 2010).

Direct electrochemistry of Shewanella oneidensis cytochrome c nitrite reductase: evidence of interactions across the dimeric interface.

Shewanella oneidensis cytochrome c nitrite reductase (soNrfA), a dimeric enzyme that houses five c-type hemes per protomer, conducts the six-electron reduction of nitrite and the two-electron reduction of hydroxylamine. Protein film voltammetry (PFV) has been used to study the cytochrome c nitrite reductase from Escherichia coli (ecNrfA) previously, revealing catalytic reduction of both nitrite and hydroxylamine substrates by ecNrfA adsorbed to a graphite electrode that is characterized by "boosts" and attenuations in activity depending on the applied potential. Here, we use PFV to investigate the catalytic properties of soNrfA during both nitrite and hydroxylamine turnover and compare those properties to the properties of ecNrfA. Distinct differences in both the electrochemical and kinetic characteristics of soNrfA are observed; e.g., all detected electron transfer steps are one-electron in nature, contrary to what has been observed in ecNrfA [Angove, H. C., Cole, J. A., Richardson, D. J., and Butt, J. N. (2002) J. Biol. Chem. 277, 23374-23381]. Additionally, we find evidence of substrate inhibition during nitrite turnover and negative cooperativity during hydroxylamine turnover, neither of which has previously been observed in any cytochrome c nitrite reductase. Collectively, these data provide evidence that during catalysis, potential pathways of communication exist between the individual soNrfA monomers comprising the native homodimer.

Reductive activation of the heme iron-nitrosyl intermediate in the reaction mechanism of cytochrome c nitrite reductase: a theoretical study.

Cytochrome c nitrite reductase catalyzes the six-electron, seven-proton reduction of nitrite to ammonia without release of any detectable reaction intermediate. This implies a unique flexibility of the active site combined with a finely tuned proton and electron delivery system. In the present work, we employed density functional theory to study the recharging of the active site with protons and electrons through the series of reaction intermediates based on nitrogen monoxide [Fe(II)-NO(+), Fe(II)-NO·, Fe(II)-NO(-), and Fe(II)-HNO]. The activation barriers for the various proton and electron transfer steps were estimated in the framework of Marcus theory. Using the barriers obtained, we simulated the kinetics of the reduction process. We found that the complex recharging process can be accomplished in two possible ways: either through two consecutive proton-coupled electron transfers (PCETs) or in the form of three consecutive elementary steps involving reduction, PCET, and protonation. Kinetic simulations revealed the recharging through two PCETs to be a means of overcoming the predicted deep energetic minimum that is calculated to occur at the stage of the Fe(II)-NO· intermediate. The radical transfer role for the active-site Tyr(218), as proposed in the literature, cannot be confirmed on the basis of our calculations. The role of the highly conserved calcium located in the direct proximity of the active site in proton delivery has also been studied. It was found to play an important role in the substrate conversion through the facilitation of the proton transfer steps.

Covalent modifications of the catalytic tyrosine in octahaem cytochrome c nitrite reductase and their effect on the enzyme activity.

Octahaem cytochrome c nitrite reductase from Thioalkalivibrio nitratireducens (TvNiR), like the previously characterized pentahaem nitrite reductases (NrfAs), catalyzes the six-electron reductions of nitrite to ammonia and of sulfite to sulfide. The active site of both TvNiR and NrfAs is formed by the lysine-coordinated haem and His, Tyr and Arg residues. The distinguishing structural feature of TvNiR is the presence of a covalent bond between the CE2 atom of the catalytic Tyr303 and the S atom of Cys305, which might be responsible for the higher nitrite reductase activity of TvNiR compared with NrfAs. In the present study, a new modified form of the enzyme (TvNiRb) that contains an additional covalent bond between Tyr303 CE1 and Gln360 CG is reported. Structures of TvNiRb in complexes with phosphate (1.45 Šresolution) and sulfite (1.8 Šresolution), the structure of TvNiR in a complex with nitrite (1.83 Šresolution) and several additional structures were determined. The formation of the second covalent bond by Tyr303 leads to a decrease in both the nitrite and sulfite reductase activities of the enzyme. Tyr303 is located at the exit from the putative proton-transport channel to the active site, which is absent in NrfAs. This is an additional argument in favour of the involvement of Tyr303 as a proton donor in catalysis. The changes in the activity of cytochrome c nitrite reductases owing to the formation of Tyr-Cys and Tyr-Gln bonds may be associated with changes in the pK(a) value of the catalytic tyrosine.

Characterization of the active site and calcium binding in cytochrome c nitrite reductases.

The decahaem homodimeric cytochrome c nitrite reductase (NrfA) is expressed within the periplasm of a wide range of Gamma-, Delta- and Epsilon-proteobacteria and is responsible for the six-electron reduction of nitrite to ammonia. This allows nitrite to be used as a terminal electron acceptor, facilitating anaerobic respiration while allowing nitrogen to remain in a biologically available form. NrfA has also been reported to reduce nitric oxide (a reaction intermediate) and sulfite to ammonia and sulfide respectively, suggesting a potential secondary role as a detoxification enzyme. The protein sequences and crystal structures of NrfA from different bacteria and the closely related octahaem nitrite reductase from Thioalkalivibrio nitratireducens (TvNir) reveal that these enzymes are homologous. The NrfA proteins contain five covalently attached haem groups, four of which are bis-histidine-co-ordinated, with the proximal histidine being provided by the highly conserved CXXCH motif. These haems are responsible for intraprotein electron transfer. The remaining haem is the site for nitrite reduction, which is ligated by a novel lysine residue provided by a CXXCK haem-binding motif. The TvNir nitrite reductase has five haems that are structurally similar to those of NrfA and three extra bis-histidine-coordinated haems that precede the NrfA conserved region. The present review compares the protein sequences and structures of NrfA and TvNir and discusses the subtle differences related to active-site architecture and Ca2+ binding that may have an impact on substrate reduction.