Recent mechanistic developments for cytochrome c nitrite reductase, the key enzyme in the dissimilatory nitrate reduction to ammonium pathway.

Cytochrome c nitrite reductase, NrfA, is a soluble, periplasmic pentaheme cytochrome responsible for the reduction of nitrite to ammonium in the Dissimilatory Nitrate Reduction to Ammonium (DNRA) pathway, a vital reaction in the global nitrogen cycle. NrfA catalyzes this six-electron and eight-proton reduction of nitrite at a single active site with the help of its quinol oxidase partners. In this review, we summarize the latest progress in elucidating the reaction mechanism of ammonia production, including new findings about the active site architecture of NrfA, as well as recent results that elucidate electron transfer and storage in the pentaheme scaffold of this enzyme.

Structural and functional insights into lysine acetylation of cytochrome c using mimetic point mutants.

Post-translational modifications frequently modulate protein functions. Lysine acetylation in particular plays a key role in interactions between respiratory cytochrome c and its metabolic partners. To date, in vivo acetylation of lysines at positions 8 and 53 has specifically been identified in mammalian cytochrome c, but little is known about the structural basis of acetylation-induced functional changes. Here, we independently replaced these two residues in recombinant human cytochrome c with glutamine to mimic lysine acetylation and then characterized the structure and function of the resulting K8Q and K53Q mutants. We found that the physicochemical features were mostly unchanged in the two acetyl-mimetic mutants, but their thermal stability was significantly altered. NMR chemical shift perturbations of the backbone amide resonances revealed local structural changes, and the thermodynamics and kinetics of electron transfer in mutants immobilized on gold electrodes showed an increase in both protein dynamics and solvent involvement in the redox process. We also observed that the K8Q (but not the K53Q) mutation slightly increased the binding affinity of cytochrome c to its physiological electron donor, cytochrome c1 -which is a component of mitochondrial complex III, or cytochrome bc1 -thus suggesting that Lys8 (but not Lys53) is located in the interaction area. Finally, the K8Q and K53Q mutants exhibited reduced efficiency as electron donors to complex IV, or cytochrome c oxidase.

Reperfusion mediates heme impairment with increased protein cysteine sulfonation of mitochondrial complex III in the post-ischemic heart.

A serious consequence of myocardial ischemia-reperfusion injury (I/R) is oxidative damage, which causes mitochondrial dysfunction. The cascading ROS can propagate and potentially induce heme bleaching and protein cysteine sulfonation (PrSO3H) of the mitochondrial electron transport chain. Herein we studied the mechanism of I/R-mediated irreversible oxidative injury of complex III in mitochondria from rat hearts subjected to 30-min of ischemia and 24-h of reperfusion in vivo. In the I/R region, the catalytic activity of complex III was significantly impaired. Spectroscopic analysis indicated that I/R mediated the destruction of hemes b and c + c1 in the mitochondria, supporting I/R-mediated complex III impairment. However, no significant impairment of complex III activity and heme damage were observed in mitochondria from the risk region of rat hearts subjected only to 30-min ischemia, despite a decreased state 3 respiration. In the I/R mitochondria, carbamidomethylated C122/C125 of cytochrome c1 via alkylating complex III with a down regulation of HCCS was exclusively detected, supporting I/R-mediated thioether defect of heme c1. LC-MS/MS analysis showed that I/R mitochondria had intensely increased complex III PrSO3H levels at the C236 ligand of the [2Fe2S] cluster of the Rieske iron­sulfur protein (uqcrfs1), thus impairing the electron transport activity. MS analysis also indicated increased PrSO3H of the hinge protein at C65 and of cytochrome c1 at C140 and C220, which are confined in the intermembrane space. MS analysis also showed that I/R extensively enhanced the PrSO3H of the core 1 (uqcrc1) and core 2 (uqcrc2) subunits in the matrix compartment, thus supporting the conclusion that complex III releases ROS to both sides of the inner membrane during reperfusion. Analysis of ischemic mitochondria indicated a modest reduction from the basal level of complex III PrSO3H detected in the mitochondria of sham control hearts, suggesting that the physiologic hyperoxygenation and ROS overproduction during reperfusion mediated the enhancement of complex III PrSO3H. In conclusion, reperfusion-mediated heme damage with increased PrSO3H controls oxidative injury to complex III and aggravates mitochondrial dysfunction in the post-ischemic heart.

A Kinetic Investigation of the Early Steps in Cytochrome c Nitrite Reductase (ccNiR)-Catalyzed Reduction of Nitrite.

The decaheme enzyme cytochrome c nitrite reductase (ccNiR) catalyzes reduction of nitrite to ammonium in a six-electron, eight-proton process. With a strong reductant as the electron source, ammonium is the sole product. However, intermediates accumulate when weaker reductants are employed, facilitating study of the ccNiR mechanism. Herein, the early stages of Shewanella oneidensis ccNiR-catalyzed nitrite reduction were investigated by using the weak reductants N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) and ferrocyanide. In stopped-flow experiments, reduction of nitrite-loaded ccNiR by TMPD generated a transient intermediate, identified as FeH1II(NO2-), where FeH1 represents the ccNiR active site. FeH1II(NO2-) accumulated rapidly and was then more slowly converted to the two-electron-reduced moiety {FeH1NO}7; ccNiR was not reduced beyond the {FeH1NO}7 state. The midpoint potentials for sequential reduction of FeH1III(NO2-) to FeH1II(NO2-) and then to {FeH1NO}7 were estimated to be 130 and 370 mV versus the standard hydrogen electrode, respectively. FeH1II(NO2-) does not accumulate at equilibrium because its reduction to {FeH1NO}7 is so much easier than the reduction of FeH1III(NO2-) to FeH1II(NO2-). With weak reductants, free NO• was released from nitrite-loaded ccNiR. The release of NO• from {FeH1NO}7 is exceedingly slow (k ∼ 0.001 s-1), but it is somewhat faster (k ∼ 0.050 s-1) while FeH1III(NO2-) is being reduced to {FeH1NO}7; then, the release of NO• from the undetectable transient {FeH1NO}6 can compete with reduction of {FeH1NO}6 to {FeH1NO}7. CcNiR appears to be optimized to capture nitrite and minimize the release of free NO•. Nitrite capture is achieved by reducing bound nitrite with even weak electron donors, while NO• release is minimized by stabilizing the substitutionally inert {FeH1NO}7 over the more labile {FeH1NO}6.

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.

Theoretical Description of the Primary Proton-Coupled Electron Transfer Reaction in the Cytochrome bc1 Complex.

The cytochrome bc1 complex is a transmembrane enzymatic protein complex that plays a central role in cellular energy production and is present in both photosynthetic and respiratory chain organelles. Its reaction mechanism is initiated by the binding of a quinol molecule to an active site, followed by a series of charge transfer reactions between the quinol and protein subunits. Previous work hypothesized that the primary reaction was a concerted proton-coupled electron transfer (PCET) reaction because of the apparent absence of intermediate states associated with single proton or electron transfer reactions. In the present study, the kinetics of the primary bc1 complex PCET reaction is investigated with a vibronically nonadiabatic PCET theory in conjunction with all-atom molecular dynamics simulations and electronic structure calculations. The computed rate constants and relatively high kinetic isotope effects are consistent with experimental measurements on related biomimetic systems. The analysis implicates a concerted PCET mechanism with significant hydrogen tunneling and nonadiabatic effects in the bc1 complex. Moreover, the employed theoretical framework is shown to serve as a general strategy for describing PCET reactions in bioenergetic systems.

The proton pumping mechanism of the bc1 complex.

The bc1 complex is a proton pump of the mitochondrial electron transport chain which transfers electrons from ubiquinol to cytochrome c. It operates via the modified Q cycle in which the two electrons from oxidation of ubiquinol at the Qo center are bifurcated such that the first electron is passed to Cytc via an iron sulfur center and c1 whereas the second electron is passed across the membrane by bL and bH to reduce ubiquinone at the Qi center. Proton pumping occurs because oxidation of ubiquinol at the Qo center releases protons to the P-side and reduction of ubiquinone at the Qi center takes up protons from the N-side. However, the mechanisms which prevent the thermodynamically more favorable short circuit reactions and so ensure precise bifurcation and proton pumping are not known. Here we use statistical thermodynamics to show that reaction steps that originate from high energy states cannot support high flux even when they have large rate constants. We show how the chemistry of ubiquinol oxidation and the structure of the Qo site can result in free energy profiles that naturally suppress flux through the short circuit pathways while allowing high rates of bifurcation. These predictions are confirmed through in-silico simulations using a Markov state model.

Physical contact between cytochrome c1 and cytochrome c increases the driving force for electron transfer.

In oxidative phosphorylation, the transfer of electrons from reduced cofactors to molecular oxygen via the electron transport chain (ETC) sustains the electrochemical transmembrane potential needed for ATP synthesis. A key component of the ETC is complex III (CIII, cytochrome bc1), which transfers electrons from reduced ubiquinone to soluble cytochrome c (Cc) coupled to proton translocation into the mitochondrial intermembrane space. One electron from every two donated by hydroquinone at site P is transferred to Cc via the Rieske-cytochrome c1 (Cc1) pathway. According to recent structural analyses of CIII and its transitory complex with Cc, the interaction between the Rieske subunit and Cc1 switches intermittently during CIII activity. However, the electrochemical properties of Cc1 and their function as a wire between Rieske and Cc are rather unexplored. Here, temperature variable cyclic voltammetry provides novel data on the thermodynamics and kinetics of interfacial electron transfer of immobilized Cc1. Findings reveal that Cc1 displays two channels for electron exchange, with a remarkably fast heterogeneous electron transfer rate. Furthermore, the electrochemical properties are strongly modulated by the binding mode of the protein. Additionally, we show that electron transfer from Cc1 to Cc is thermodynamically favored in the immobilized Cc1-Cc complex. Nuclear Magnetic Resonance, HADDOCK, and Surface Plasmon Resonance experiments provide further structural and functional data of the Cc1-Cc complex. Our data supports the Rieske-Cc1-Cc pathway acting as a unilateral switch thyristor in which redox potential modulation through protein-protein contacts are complemented with the relay-like Rieske behavior.

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.

Mutational analysis of the Qi-site proton pathway in yeast cytochrome bc1 complex.

The respiratory cytochrome bc1 complex functions as a protonmotive ubiquinol:cytochrome c oxidoreductase. Lysine 228 (K228) located within the quinol reduction (Qi) site of the bc1 complex, has been reported as a key residue for proton transfer during the redox chemistry cycle to substrate quinone at Qi. In yeast, while single mutations had no effect, the combination of K228L and F225L resulted in a severe respiratory growth defect and inhibition of O2 consumption in intact cells. The inhibition was overcome by uncoupling the mitochondrial membrane or by suppressor mutations in the region of K228L-F225L. We propose that the K228L mutation introduces energetic (and kinetic) barriers into normal electron- and proton transfer chemistry at Qi, which are relieved by dissipation of the opposing protonmotive force or through the restoration of favourable intraprotein proton transfer networks via suppressor mutation.

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.

Monitoring alkaline transitions of yeast iso-1 cytochrome c at natural isotopic abundance using trimethyllysine as a native NMR probe.

Spectral overlap makes it difficult to use NMR for mapping the conformational profile of heterogeneous conformational ensembles of macromolecules. Here, we apply a 1H-14N HSQC experiment to monitor the alkaline conformational transitions of yeast iso-1 cytochrome c (ycyt c) at natural isotopic abundance. Trimethylated Lys72 of ycyt c is selectively detected by a 1H-14N HSQC experiment, and used as a probe to trace conformational transitions of ycyt c under alkaline conditions. It was found that at least four different conformers of ycyt c coexisted under alkaline conditions. Besides the native structure, Lys73 or Lys79 coordinated conformers and a partially unfolded state with exposed heme were observed. These results indicate that the method is powerful at simplifying spectra of a trimethylated protein, which makes it possible to study complex conformational transitions of naturally extracted or chemically modified trimethylated protein at natural isotopic abundance.

Functional flexibility of electron flow between quinol oxidation Qo site of cytochrome bc1 and cytochrome c revealed by combinatory effects of mutations in cytochrome b, iron-sulfur protein and cytochrome c1.

Transfer of electron from quinol to cytochrome c is an integral part of catalytic cycle of cytochrome bc1. It is a multi-step reaction involving: i) electron transfer from quinol bound at the catalytic Qo site to the Rieske iron-sulfur ([2Fe-2S]) cluster, ii) large-scale movement of a domain containing [2Fe-2S] cluster (ISP-HD) towards cytochrome c1, iii) reduction of cytochrome c1 by reduced [2Fe-2S] cluster, iv) reduction of cytochrome c by cytochrome c1. In this work, to examine this multi-step reaction we introduced various types of barriers for electron transfer within the chain of [2Fe-2S] cluster, cytochrome c1 and cytochrome c. The barriers included: impediment in the motion of ISP-HD, uphill electron transfer from [2Fe-2S] cluster to heme c1 of cytochrome c1, and impediment in the catalytic quinol oxidation. The barriers were introduced separately or in various combinations and their effects on enzymatic activity of cytochrome bc1 were compared. This analysis revealed significant degree of functional flexibility allowing the cofactor chains to accommodate certain structural and/or redox potential changes without losing overall electron and proton transfers capabilities. In some cases inhibitory effects compensated one another to improve/restore the function. The results support an equilibrium model in which a random oscillation of ISP-HD between the Qo site and cytochrome c1 helps maintaining redox equilibrium between all cofactors of the chain. We propose a new concept in which independence of the dynamics of the Qo site substrate and the motion of ISP-HD is one of the elements supporting this equilibrium and also is a potential factor limiting the overall catalytic rate.

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.

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.

Construction of effective disposable biosensors for point of care testing of nitrite.

In this paper we aim to demonstrate, as a proof-of-concept, the feasibility of the mass production of effective point of care tests for nitrite quantification in environmental, food and clinical samples. Following our previous work on the development of third generation electrochemical biosensors based on the ammonia forming nitrite reductase (ccNiR), herein we reduced the size of the electrodes' system to a miniaturized format, solved the problem of oxygen interference and performed simple quantification assays in real samples. In particular, carbon paste screen printed electrodes (SPE) were coated with a ccNiR/carbon ink composite homogenized in organic solvents and cured at low temperatures. The biocompatibility of these chemical and thermal treatments was evaluated by cyclic voltammetry showing that the catalytic performance was higher with the combination acetone and a 40°C curing temperature. The successful incorporation of the protein in the carbon ink/solvent composite, while remaining catalytically competent, attests for ccNiR's robustness and suitability for application in screen printed based biosensors. Because the direct electrochemical reduction of molecular oxygen occurs when electroanalytical measurements are performed at the negative potentials required to activate ccNiR (ca.-0.4V vs Ag/AgCl), an oxygen scavenging system based on the coupling of glucose oxidase and catalase activities was successfully used. This enabled the quantification of nitrite in different samples (milk, water, plasma and urine) in a straightforward way and with small error (1-6%). The sensitivity of the biosensor towards nitrite reduction under optimized conditions was 0.55 A M(-1) cm(-2) with a linear response range 0.7-370 µM.

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.

Molecular Study of the G1 Haplotypes of Echinococcus granulosus from Iran Based on Cytochrome C Oxidase (Subunit 1) Sequence.

OBJECTIVE: In this study, we attempted to identify new Echinococcus granulosus isolates in the North West provinces of Iran based on the mitochondrial cytochrome c oxidase subunit 1 (CO1) sequence. METHODS: Twenty-nine hydatid cysts from sheep and goats were collected. Genomic DNAs were extracted, and a partial sequence of the CO1 gene was amplified. Polymerase chain reaction products were cloned and sequenced with M13 primers in both directions. RESULTS: All Iranian isolates were located in G1 and G3 genotypes. For the first time, a new G1 haplotype in two Iranian isolates were identified. CONCLUSION: It seems that this new haplotype was transmitted from Jordan to Iran or vice versa.