Search

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.

Hydroxylamine (NH2OH or HA) is a redox-active nitrogen oxide that occurs as a toxic intermediate in the oxidation of ammonium by nitrifying and methanotrophic bacteria. Within ammonium containing environments, HA is generated by ammonia monooxygenase (nitrifiers) or methane monooxygenase (methanotrophs). Subsequent oxidation of HA is catalyzed by heme proteins, including cytochromes P460 and multiheme hydroxylamine oxidoreductases, the former contributing to emissions of N2O, an ozone-depleting greenhouse gas. A heme-HA complex is also a proposed intermediate in the reduction of nitrite to ammonia by cytochrome c nitrite reductase. Despite the importance of heme-HA complexes within the biogeochemical nitrogen cycle, fundamental aspects of their coordination chemistry remain unknown, including the effect of the Fe redox state on heme-HA affinity, kinetics, and spectroscopy. Using stopped-flow UV-vis and resonance Raman spectroscopy, we investigated HA complexes of the L16G distal pocket variant of Alcaligenes xylosoxidans cytochrome c'-α (L16G AxCP-α), a pentacoordinate c-type cytochrome that we show binds HA in its Fe(III) (Kd ∼ 2.5 mM) and Fe(II) (Kd = 0.0345 mM) states. The ∼70-fold higher HA affinity of the Fe(II) state is due mostly to its lower koff value (0.0994 s-1 vs 11 s-1), whereas kon values for Fe(II) (2880 M-1 s-1) and Fe(III) (4300 M-1 s-1) redox states are relatively similar. A comparison of the HA and imidazole affinities of L16G AxCP-α was also used to predict the influence of Fe redox state on HA binding to other proteins. Although HA complexes of L16G AxCP-α decompose via redox reactions, the lifetime of the Fe(II)HA complex was prolonged in the presence of excess reductant. Spectroscopic parameters determined for the Fe(II)HA complex include the N-O stretching vibration of the NH2OH ligand, ν(N-O) = 906 cm-1. Overall, the kinetic trends and spectroscopic benchmarks from this study provide a foundation for future investigations of heme-HA reaction mechanisms.

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.

The present thesis focuses on the application of two distinct enzymes – cytochrome c nitrite reductase (ccNiR) and paraoxonase 1 (PON1) – as the key elements in the development of two electrochemical analytical devices, namely a 3rd generation miniaturized biosensor for nitrite quantification, and a 1st generation biosensor for the detection of homocysteine thiolactone (HTL). The analytical surveillance of nitrite is crucial in the management of public health and environmental safety. Additionally, it is considered an important marker of proper endothelial function and a common indicator of urinary tract infection. The endogenously produced HTL has been implied in the pathophysiology of the vascular system, making the detection and quantification of this metabolite necessary in the study and diagnosis of processes related with cardiovascular diseases. The biocompatibility of ccNiR with carbon conductive inks and low-temperature curing processes was previously demonstrated on standard pyrolytic graphite electrodes. In this work, the next step in the development of a disposable miniaturized nitrite biosensor was performed, by manually applying a carbon ink and ccNiR composite on carbon-screen printed electrodes. The analytical performance was evaluated by cyclic voltammetry, and the bioelectrodes obtained in this way showed good catalytic response towards nitrite, although with poor reproducibility. Future automated fabrication processes could improve this. Nonetheless, the successful incorporation of the catalytically competent and stable enzyme, indicate a putative application for future co-printing with the transducing electrode. Due to the need of an oxygen-free environment, two enzymatic deoxygenation systems were integrated with the transducer: one based on the well-known couple glucose oxidase/catalase, while the other was a novel system based on multicopper oxidases. Overall, the combination of these enzymes with ccNiR on carbon screen-printed electrodes resulted in miniaturized nitrite voltammetric biosensors capable of operating at low potentials, under ambient air. Additionally, for the measurement of nitrite dynamics in cerebral tissue, carbon fiber microelectrodes and ceramic-based platinum microelectrodes arrays were employed as transducers in the development of microscale (bio)sensing platforms. Preliminary data showed that exogenous nitrite real-time clearance could thus be detected in situ and in vivo. Owing to the putative relation between lower PON1 activity and the greater risk of developing diseases with an inflammatory component, a novel electrochemical protocol for PON1 status determination was developed. Additionally, the potential role of the enzyme as the bioreceptor in an HTL voltammetric biosensor was also addressed.

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

A cobalt-tripeptide complex (CoGGH) is developed as an electrocatalyst for the selective six-electron, eight-proton reduction of nitrite to ammonium in aqueous buffer near neutral pH. The onset potential for nitrite reduction occurs at -0.65 V vs Ag/AgCl (1 M KCl). Controlled potential electrolysis at -0.90 V generates ammonium with a faradaic efficiency of 90 ± 3% and a turnover number of 3550 ± 420 over 5.5 h. CoGGH also catalyzes the reduction of the proposed intermediates nitric oxide and hydroxylamine to ammonium. These results reveal that a simple metallopeptide is an active functional mimic of the complex enzymes cytochrome c nitrite reductase and siroheme-containing nitrite reductase.

The structure of cytochrome c nitrite reductase from the bacterium Thioalkalivibrio nitratireducens was determined by cryo-electron microscopy (cryo-EM) at a 2.56 resolution. Possible structural heterogeneity of the enzyme was assessed. The backbone and side-chain orientations in the cryo-EM-based model are, in general, similar to those in the high-resolution X-ray diffraction structure of this enzyme.

The fundamental biogeochemical cycle of nitrogen includes cytochrome c nitrite reductase, which catalyzes the reduction of nitrite ions to ammonium with eight protons and six electrons (NO2– + 8H+ + 6e– → NH4+ + 2H2O). This reaction has motivated researchers to explore the reduction of nitrite. Although a number of electrochemical reductions of NO2– have been reported, the synthetic nitrite reduction reaction remains limited. To the best of our knowledge, formation of ammonia has not been reported. We report a three-step nitrite reduction cycle on a dinuclear ruthenium platform {(TpRu)2(μ-pz)} (Tp = HB(pyrazol-1-yl)3), producing ammonia. The cycle comprises conversion of a nitrito ligand to a NO ligand using 2H+ and e–, subsequent reduction of the NO ligand to a nitrido and a H2O ligand by consumption of 2H+ and 5e–, and recovery of the parent nitrito ligand. Moreover, release of ammonia was detected.

The present study reports a novel voltammetric biosensor for cyanide based on its inhibitory effect on cytochrome c nitrite reductase (ccNiR). Interestingly, the earlier development of a point-of-care test for nitrite based on the direct electrochemistry of ccNiR has shown that the cyanide inhibition depends on the type of carbon material employed as transducer ( Monteiro et al., 2019 ). In this work, commercial graphite pencil leads were employed in the construction of both working and pseudo-reference electrodes, with ccNiR being simply drop casted onto the former. In this way, we produced a functional and fully integrated voltammetric biosensor for nitrite quantification that also allows to observe a decrease in the catalytic current due to cyanide addition. Under turnover conditions, the biosensor showed a linear response with the logarithm of cyanide concentration in the 5–76 μM (cyclic voltammetry) and 1–40 μM (square-wave voltammetry) ranges, with a sensitivity of 20–25% ln [cyanide μM]−1 and a detection limit of 0.86–4.4 μM. The application of the pencil lead as a putative pseudo-reference was very promising, since the potentials profile matched those observed with a true reference electrode (Ag/AgCl). Overall, the direct electron transfer between ccNiR and a pencil lead electrode was demonstrated for the first time, with cyanide-induced inhibition being easily monitored, paving the way for the employment of these low-cost bioelectrodes as cyanide probes for on-site surveillance of aquatic environments.

As bridge in global cycles of carbon, nitrogen, and sulfur, sulfate-reducing bacteria (SRB) play more and more important role under various environments, especially the saline-alkali environments with significant increase in area caused by human activities. Sulfate reduction can be inhibited by environmental nitrate. However, how SRB cope with environmental nitrate stress in these extreme environments still remain unclear. Here, after a long-term enrichment of sediment from saline-alkali Qinghai Lake of China using anaerobic filter reactors, nitrate was added to evaluate the response of SRB. With the increase in nitrate concentrations, the inhibition on sulfate reduction was gradually observed. Interestingly, extension of hydraulic retention time can relieve the inhibition caused by high nitrate concentration. Mass balance analysis showed that nitrate reduction is prior to sulfate reduction. Further metatranscriptomic analysis shows that, genes of nitrite reductase (periplasmic cytochrome c nitrite reductase gene) and energy metabolisms (lactate dehydrogenase, formate dehydrogenase, pyruvate:ferredoxin-oxidoreductase, and fumarate reductase genes) in SRB was down-regulated, challenging the long-held opinion that up-regulation of these genes can relieve the nitrate inhibition. Most importantly, the nitrate addition activated the denitrification pathway in denitrifying bacteria (DB) via significantly up-regulating the expression of the corresponding genes (nitrite reductase, nitric oxide reductase c subunit, nitric oxide reductase activation protein and nitrous oxide reductase genes), quickly reducing the environmental nitrate and relieving the nitrate inhibition on SRB. Our findings unravel that in response to environmental nitrate stress, haloalkaliphilic SRB show dependency on DB, and expand our knowledge of microbial relationship during sulfur and nitrogen cycles.

This work was supported by the Applied Molecular Biosciences Unit-UCIBIO which is financed by national funds from FCT/MCTES (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728). C.M.S. and T.M. thank the financial support from Fundacao para a Ciencia e Tecnologia (Fellowships SFRH/BPD/79566/2011 and PD/BD/109687/2015). The ubiquitous nitrite is a major analyte in the management of human health and environmental risks. The current analytical methods are complex techniques that do not fulfil the need for simple, robust and low-cost tools for on-site monitoring. Electrochemical reductase-based biosensors are presented as a powerful alternative, due to their good analytical performance and miniaturization potential. However, their real-world application is limited by the need of anoxic working conditions, and the standard oxygen removal strategies are incompatible with point-of-care measurements. Instead, a bienzymatic oxygen scavenger system comprising glucose oxidase and catalase can be used to promote anoxic conditions in aired environments. Herein, carbon screen-printed electrodes were modified with cytochrome c nitrite reductase together with glucose oxidase and catalase, so that nitrite cathodic detection could be performed by cyclic voltammetry under ambient air. The resulting biosensor displayed good linear response to the analyte (2–200 µM, sensitivity of 326 ± 5 mA M −1 cm −2 at −0.8 V; 0.8–150 µM, sensitivity of 511 ± 11 mA M −1 cm −2 at −0.5 V), while being free from oxygen interference and stable up to 1 month. Furthermore, the biosensor’s catalytic response was unaffected by the presence of cyanide, a well-known inhibitor of heme-enzymes. publishersversion published

Direct electron transfer with redox proteins, in third generation biosensors, is already proved to be favored on electrodes modified with nanoparticles. In this work, different crystallographic and morphologic structures of tungsten oxide (WO3) nanoparticles are modified by hydrothermal synthesis at 180 °C. The electrochemical properties of WO3 nanoparticles deposit on ITO electrodes are investigated and the analytical performance of the nitrite biosensor is presented as proof of concept. Despite the inherent features of each nanostructure, the heterogeneous electron transfer with the WO3 nanoparticles modified electrodes is thoroughly improved and, very importantly, the cytochrome c nitrite reductase (ccNiR) enzyme is able to keep its biological function. When compared with bare commercial ITO electrodes, the exchange rate constant of WO3/ITO electrodes with cytochrome c increased one order of magnitude, while the analytical parameters of the ccNiR/WO3/ITO electrodes response to nitrite (the Michaelis–Menten constant is 47 μM and sensitivity of 2143 mA M −1 cm−2) are comparable to those reported for carbon based electrodes. Therefore, these metal oxide nanoparticles are good alternative materials for electrochemical applications, such as non-mediated biosensors.

Sensing potential nitrogen-containing respiratory substrates such as nitrate, nitrite, hydroxylamine, nitric oxide (NO) or nitrous oxide (N2 O) in the environment and subsequent upregulation of corresponding catabolic enzymes is essential for many microbial cells. The molecular mechanisms of such adaptive responses are, however, highly diverse in different species. Here, induction of periplasmic nitrate reductase (Nap), cytochrome c nitrite reductase (Nrf) and cytochrome c N2 O reductase (cNos) was investigated in cells of the Epsilonproteobacterium Wolinella succinogenes grown either by fumarate, nitrate or N2 O respiration. Furthermore, fumarate respiration in the presence of various nitrogen compounds or NO-releasing chemicals was examined. Upregulation of each of the Nap, Nrf and cNos enzyme systems was found in response to the presence of nitrate, NO-releasers or N2 O, and the cells were shown to employ three transcription regulators of the Crp-Fnr superfamily (homologues of Campylobacter jejuni NssR), designated NssA, NssB and NssC, to mediate the upregulation of Nap, Nrf and cNos. Analysis of single nss mutants revealed that NssA controls production of the Nap and Nrf systems in fumarate-grown cells, while NssB was required to induce the Nap, Nrf and cNos systems specifically in response to NO-generators. NssC was indispensable for cNos production under any tested condition. The data indicate dedicated signal transduction routes responsive to nitrate, NO and N2 O and imply the presence of an N2 O-sensing mechanism.

Octahaem cytochromecnitrite reductase from the bacteriumThioalkalivibrio nitratireducenscatalyzes 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 cytochromecnitrite 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.

© 2017 Kublanov, Sigalova, Gavrilov, Lebedinsky, Rinke, Kovaleva, Chernyh, Ivanova, Daum, Reddy, Klenk, Spring, Göker, Reva, Miroshnichenko, Kyrpides, Woyke, Gelfand, Bonch-Osmolovskaya. The genome of Caldithrix abyssi, the first cultivated representative of a phylum-level bacterial lineage, was sequenced within the framework of Genomic Encyclopedia of Bacteria and Archaea (GEBA) project. The genomic analysis revealed mechanisms allowing this anaerobic bacterium to ferment peptides or to implement nitrate reduction with acetate or molecular hydrogen as electron donors. The genome encoded five different [NiFe]- and [FeFe]-hydrogenases, one of which, group 1 [NiFe]-hydrogenase, is presumably involved in lithoheterotrophic growth, three other produce H2during fermentation, and one is apparently bidirectional. The ability to reduce nitrate is determined by a nitrate reductase of the Nap family, while nitrite reduction to ammonia is presumably catalyzed by an octaheme cytochrome c nitrite reductase eHao. The genome contained genes of respiratory polysulfide/thiosulfate reductase, however, elemental sulfur and thiosulfate were not used as the electron acceptors for anaerobic respiration with acetate or H2, probably due to the lack of the gene of the maturation protein. Nevertheless, elemental sulfur and thiosulfate stimulated growth on fermentable substrates (peptides), being reduced to sulfide, most probably through the action of the cytoplasmic sulfide dehydrogenase and/or NAD(P)-dependent [NiFe]-hydrogenase (sulfhydrogenase) encoded by the genome. Surprisingly, the genome of this anaerobic microorganism encoded all genes for cytochrome c oxidase, however, its maturation machinery seems to be non-operational due to genomic rearrangements of supplementary genes. Despite the fact that sugars were not among the substrates reported when C. abyssi was first described, our genomic analysis revealed multiple genes of glycoside hydrolases, and some of them were predicted to be secreted. This finding aided in bringing out four carbohydrates that supported the growth of C. abyssi: starch, cellobiose, glucomannan and xyloglucan. The genomic analysis demonstrated the ability of C. abyssi to synthesize nucleotides and most amino acids and vitamins. Finally, the genomic sequence allowed us to perform a phylogenomic analysis, based on 38 protein sequences, which confirmed the deep branching of this lineage and justified the proposal of a novel phylum Calditrichaeota.

Investigations of reduction of nitrite (NO2–) to ammonia (NH3) by nitrogenase indicate a limiting stoichiometry, NO2– + 6e– + 12ATP + 7H+ → NH3 + 2H2O + 12ADP + 12Pi. Two intermediates freeze-trapped during NO2– turnover by nitrogenase variants and investigated by Q-band ENDOR/ESEEM are identical to states, denoted H and I, formed on the pathway of N2 reduction. The proposed NO2– reduction intermediate hydroxylamine (NH2OH) is a nitrogenase substrate for which the H and I reduction intermediates also can be trapped. Viewing N2 and NO2– reductions in light of their common reduction intermediates and of NO2– reduction by multiheme cytochrome c nitrite reductase (ccNIR) leads us to propose that NO2– reduction by nitrogenase begins with the generation of NO2H bound to a state in which the active-site FeMo-co (M) has accumulated two [e–/H+] (E2), stored as a (bridging) hydride and proton. Proton transfer to NO2H and H2O loss leaves M–[NO+]; transfer of the E2 hydride to the [NO+] directly to form HNO bound to ...

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.

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).

NOTICE: this is the author’s version of a work that was accepted for publication in Electrochemistry Communications. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Electrochemistry Communications, VOL. 35, (October 2013). http://dx.doi.org/10.1016/j.elecom.2013.07.027 "This work reports the evaluation of a set of multi-walled carbon nanotubes (MWCNT) presenting different surface chemistries, as interfaces for the direct electrochemistry of the multihemic nitrite reductase (ccNiR) from Desulfovibrio desulfuricans ATCC27774 (Dd). The carbon nanotubes dispersions were prepared in aqueous media and deposited on pyrolytic graphite (PG) macroelectrodes, following a layer-by-layer methodology. The resulting MWCNT bed was coated with ccNiR and studied by cyclic voltammetry. Interestingly, although small non-catalytic cathodic waves were detected in all carbon nanotubes bioconjugates, the complexity of these electrochemical signals was partially deconvoluted in some materials, the less acidic ones emphasizing the contribution of the catalytic centre. Consistently, these MWCNT were the most favourable for enzyme catalysis, highlighting the importance of the surface oxide functionalities to enzyme reactivity."

Shewanella oneidensis cytochrome c nitrite reductase (soNrfA), a dimeric enzyme that houses five c-type hemes per protomer, carries out 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 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) Protein film voltammetry reveals distinctive fingerprints of nitrite and hydroxylamine reduction by a cytochrome C nitrite reductase, 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 have 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.

and glyoxal oxidase, iron-dependent cysteine dioxygenase (CDO), sirohemeand [Fe4S4]-dependent sulfite reductase [3] (NirA), and T. nitratireducens cytochrome c nitrite reductase (TvNiR) (Supporting Information, Figures S4–S7). In all of the aforementioned enzymes, a covalent bond is formed autocatalytically between the C3 ring carbon atom of a tyrosine residue and the Sg atom of a neighboring cysteine residue, without requiring exogenous proteins (Scheme 1A). Because of the fascinating chemistry and potential industrial applications of these enzymes, the functional significance of the essential Tyr-Cys cofactor has been intensively investigated by synthetic chemists, physical chemists, enzymologists, and structure biologists, and tremendous progress has been made in this area. While the presence of the “home-made” cofactor TyrCys is firmly established in GO, CDO, NirA, and TvNiR, its exact role in metalloenzyme function is still not fully understood. A major limitation of our understanding is that such a post-translational modification (PTM) cannot be directly probed through traditional site-directed mutagenesis methods. To overcome this limitation, synthetic model compounds have been made, and studies of these model compounds have suggested that the cross-link between the Tyr and Cys residues significantly decreases the pKa value and reduction potential of the phenol side chain, and therefore facilitates proton-coupled electron transfer (PCET) between the substrate and the Tyr-Cys cofactor, which is critical for optimizing enzyme activity. Despite these advances, questions about the roles of the Tyr-Cys cross-link still remain, because no study has been able to directly probe the functions of the thioether substitution at the tyrosine residue without introducing extra mutations. While mutation of the cysteine residue that takes part in the Tyr-Cys cofactor formation invariably leads to disruption of the Tyr-Cys crosslink and significant decrease of enzyme activity, the extent to which the thioether substitution participates in the catalytic process has not been determined, and the possibility of a purely structural role for the Tyr-Cys cross-link has been proposed. To answer the above questions and to gain new insights into the Tyr-Cys cofactor function, we report herein the genetic incorporation of a new unnatural amino acid (UAA) 2-amino-3-(4-hydroxy-3-(methylthio)phenyl)propanoic acid 1 (hereafter termed MtTyr; Figure 1), which mimics the TyrCys cofactor, into a functional model of TvNiR in sperm whale myoglobin (Mb). This enzyme model with the incorporated UAA MtTyr was termed MtTyrMb. We then compared the activity of MtTyrMb with a mutant that has a Tyr residue at the same position in the same protein (termed TyrMb). Our Scheme 1. A) The autocatalytic post-translational modification that yields the Tyr-Cys cofactor in various metalloenzymes, including GO, CDO, NirA, and TvNiR. B) A biosynthetic route to MtTyr 1, catalyzed by the TPL mutant F36L.

A genomic island (GEI) of the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough, found to be able to migrate between two tRNA-Met loci of the genome, contains genes for rubredoxin:oxygen oxidoreductase-1 (roo1) and hybrid cluster protein-1 (hcp1) with additional copies for these genes (roo2 and hcp2) being found elsewhere on the chromosome. A suite of mutants was created in which roo2 and/or hcp2 and/or the GEI were either present or missing. The GEI and roo2 increased survival under microaerobic conditions and allowed growth in closer proximity to the air-water interface of soft agar tubes, two properties which appeared to be closely linked. When Hcp2(+) GEI(+) or Hcp2(-) GEI(+) cells, harbouring cytochrome c nitrite reductase (NrfHA) and growing on lactate and sulfate, were amended with 10 mM nitrite at mid-log phase (8-10 mM sulfide), all nitrite was reduced within 30 h with a rate of 3.0 mmol (g biomass)(-1) h(-1) after which sulfate reduction resumed. However, Hcp2(+) GEI(-) or Hcp2(-) GEI(-) cells were unable to use lactate, causing sulfide to be used as electron donor for nitrite reduction at a sixfold lower rate. Complementation studies indicated that hcp1, not roo1, enhanced the rate of nitrite reduction under these conditions. Hcp2 enhanced the rate of nitrite reduction when, in addition to lactate, hydrogen was also present as an electron donor. These results indicate a critical role of Hcps in alleviating nitrite stress in D. vulgaris Hildenborough by maintaining the integrity of electron transport chains from lactate or H(2) to NrfHA through removal of reactive nitrogen species. It thus appears that the GEI contributes considerably to the fitness of the organism, allowing improved growth in microaerobic environments found in sulfide-oxygen gradients and in environments, containing both sulfide and nitrite, through the action of Roo1 and Hcp1 respectively.

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.

Microorganisms employ diverse mechanisms to withstand physiological stress conditions exerted by reactive or toxic oxygen and nitrogen species such as hydrogen peroxide, organic hydroperoxides, superoxide anions, nitrite, hydroxylamine, nitric oxide or NO-generating compounds. This study identified components of the oxidative and nitrosative stress defence network of Wolinella succinogenes, an exceptional Epsilonproteobacterium that lacks both catalase and haemoglobins. Various gene deletion-insertion mutants were constructed, grown by either fumarate respiration or respiratory nitrate ammonification and subjected to disc diffusion, growth and viability assays under stress conditions. It was demonstrated that mainly two periplasmic multihaem c-type cytochromes, namely cytochrome c peroxidase and cytochrome c nitrite reductase (NrfA), mediated resistance to hydrogen peroxide. Two AhpC-type peroxiredoxin isoenzymes were shown to be involved in protection against different organic hydroperoxides. The phenotypes of two superoxide dismutase mutants lacking either SodB or SodB2 implied that both isoenzymes play important roles in oxygen and superoxide stress defence although they are predicted to reside in the cytoplasm and periplasm respectively. NrfA and a cytoplasmic flavodiiron protein (Fdp) were identified as key components of nitric oxide detoxification. In addition, NrfA (but not the hybrid cluster protein Hcp) was found to mediate resistance to hydroxylamine stress. The results indicate the presence of a robust oxidative and nitrosative stress defence network and identify NrfA as a multifunctional cytochrome c involved in both anaerobic respiration and stress protection.

The cytochrome c nitrite reductase NrfA is a 53 kDa pentahaem enzyme that crystallizes as a decahaem homodimer. NrfA catalyses the reduction of NO 2 − to NH 4 + through a six electron reduction pathway that is of major physiological significance to the anaerobic metabolism of enteric and sulfate reducing bacteria. NrfA receives electrons from the 21 kDa pentahaem NrfB donor protein. This requires that redox complexes form between the NrfA and NrfB pentahaem cytochromes. The formation of these complexes can be monitored using a range of methodologies for studying protein–protein interactions, including dynamic light scattering, gel filtration, analytical ultracentrifugation and visible spectroscopy. These methods have been used to show that oxidized NrfA exists in dynamic monomer–dimer equilibrium with a K d (dissociation constant) of 4 μM. Significantly, the monomeric and dimeric forms of NrfA are equally active for either the six electron reduction of NO 2 − or HSO 3 − . When mixed together, NrfA and NrfB exist in equilibrium with NrfAB, which is described by a K d of 50 nM. Thus, since NrfA and NrfB are present in micromolar concentrations in the periplasmic compartment, it is likely that NrfB remains tightly associated with its NrfA redox partner under physiological conditions.

Cytochrome c nitrite reductase is a homodimeric enzyme, containing five covalently attached c-type hemes per subunit. Four of the heme irons are bishistidine-ligated, whereas the fifth, the active site of the protein, has an unusual lysine coordination and calcium site nearby. A fascinating feature of this enzyme is that the full six-electron reduction of the nitrite is achieved without release of any detectable reaction intermediate. Moreover, the enzyme is known to work over a wide pH range. Both findings suggest a unique flexibility of the active site in the complicated six-electron, seven-proton reduction process. In the present work, we employed density functional theory to study the energetics and kinetics of the initial stages of nitrite reduction. The possible role of second-sphere active-site amino acids as proton donors was investigated by taking different possible protonation states and geometrical conformations into account. It was found that the most probable proton donor is His(277), whose spatial orientation and fine-tuned acidity lead to energetically feasible, low-barrier protonation reactions. However, substrate protonation may also be accomplished by Arg(114). The calculated barriers for this pathway are only slightly higher than the experimentally determined value of 15.2 kcal/mol for the rate-limiting step. Hence, having proton-donating side chains of different acidity within the active site may increase the operational pH range of the enzyme. Interestingly, Tyr(218), which was proposed to play an important role in the overall mechanism, appears not to take part in the reaction during the initial stage.

Microbial processes are crucial for ecosystem maintenance, yet documentation of these processes in complex open field sites is challenging. Here we used a multidisciplinary strategy (site geochemistry, laboratory biodegradation assays, and field extraction of molecular biomarkers) to deduce an ongoing linkage between aromatic hydrocarbon biodegradation and nitrogen cycling in a contaminated subsurface site. Three site wells were monitored over a 10-month period, which revealed fluctuating concentrations of nitrate, ammonia, sulfate, sulfide, methane, and other constituents. Biodegradation assays performed under multiple redox conditions indicated that naphthalene metabolism was favored under aerobic conditions. To explore in situ field processes, we measured metabolites of anaerobic naphthalene metabolism and expressed mRNA transcripts selected to document aerobic and anaerobic microbial transformations of ammonia, nitrate, and methylated aromatic contaminants. Gas chromatography-mass spectrometry detection of two carboxylated naphthalene metabolites and transcribed benzylsuccinate synthase, cytochrome c nitrite reductase, and ammonia monooxygenase genes indicated that anaerobic metabolism of aromatic compounds and both dissimilatory nitrate reduction to ammonia (DNRA) and nitrification occurred in situ . These data link formation (via DNRA) and destruction (via nitrification) of ammonia to in situ cycling of nitrogen in this subsurface habitat, where metabolism of aromatic pollutants has led to accumulation of reduced metabolic end products (e.g., ammonia and methane).

In this paper we propose the construction of a new non-mediated electrochemical biosensor for nitrite determination in complex samples. The device is based on the stable and selective cytochrome c nitrite reductase (ccNiR) from Desulfovibrio desulfuricans, which has both high turnover and heterogeneous electron transfer rates. In opposition to previous efforts making use of several redox mediators, in this work we exploited the capacity of ccNiR to display a direct electrochemical response when interacting with pyrolytic graphite (PG) surfaces. To enable the analytical application of such bioelectrode the protein was successfully incorporated within a porous silica glass made by the sol-gel process. In the presence of nitrite, the ccNiR/sol-gel/PG electrode promptly displays catalytic currents indicating that the entrapped ccNiR molecules are reduced via direct electron transfer. This result is noteworthy since the protein molecules are caged inside a non-conductive silica network, in the absence of any mediator species or electron relay. At optimal conditions, the minimum detectable concentration is 120 nM. The biosensor sensitivity is 430 mA M(-1) cm(-2) within a linear range of 0.25-50 microM, keeping a stable response up to two weeks. The analysis of nitrites in freshwaters using the method of standard addition was highly accurated.