Diversity and evolution of nitric oxide reduction in bacteria and archaea.

Nitrous oxide is a potent greenhouse gas whose production is catalyzed by nitric oxide reductase (NOR) members of the heme-copper oxidoreductase (HCO) enzyme superfamily. We identified several previously uncharacterized HCO families, four of which (eNOR, sNOR, gNOR, and nNOR) appear to perform NO reduction. These families have novel active-site structures and several have conserved proton channels, suggesting that they might be able to couple NO reduction to energy conservation. We isolated and biochemically characterized a member of the eNOR family from the bacterium Rhodothermus marinus and found that it performs NO reduction. These recently identified NORs exhibited broad phylogenetic and environmental distributions, greatly expanding the diversity of microbes in nature capable of NO reduction. Phylogenetic analyses further demonstrated that NORs evolved multiple times independently from oxygen reductases, supporting the view that complete denitrification evolved after aerobic respiration.

Computational Exploration of Minimum Energy Reaction Pathway of N2O Formation from Intermediate I of P450nor Using an Active Center Model.

P450nor is a heme-containing enzyme that catalyzes the conversion of nitric oxide (NO) to nitrous oxide (N2O). Its catalytic mechanism has attracted attention in chemistry, biology, and environmental engineering. The catalytic cycle of P450nor is proposed to consist of three major steps. The reaction mechanism for the last step, N2O generation, remains unknown. In this study, the reaction pathway of the N2O generation from the intermediate I was explored with the B3LYP calculations using an active center model after the examination of the validity of the model. In the validation, we compared the heme distortions between P450nor and other oxidoreductases, suggesting a small effect of protein environment on the N2O generation reaction in P450nor. We then evaluated the electrostatic environment effect of P450nor on the hydride affinity to the active site with quantum mechanics/molecular mechanics (QM/MM) calculations, confirming that the affinity was unchanged with or without the protein environment. The active center model for P450nor showed that the N2O generation process in the enzymatic reaction undergoes a reasonable barrier height without protein environment. Consequently, our findings strongly suggest that the N2O generation reaction from the intermediate I depends sorely on the intrinsic reactivity of the heme cofactor bound on cysteine residue.

Brucella spp. are facultative anaerobic bacteria under denitrifying conditions.

IMPORTANCE: Respiration is a fundamental and complex process that bacteria use to produce energy. Despite aerobic respiration being the most common, some bacteria make use of a mode of respiration in the absence of oxygen, called anaerobic respiration, which can yield advantages in adaptation to various environmental conditions. Denitrification is part of this respiratory process ensuring higher respiratory flexibility under oxygen depletion. Here, we report for the first time the evidence of anaerobic growth of Brucella spp. under denitrifying conditions, which implies that this genus should be reconsidered as facultative anaerobic. Our study further describes that efficient denitrification is not equally found within the Brucella genus, with atypical species showing a greater ability to denitrify, correlated with higher expression of the genes involved, as compared to classical species.

The copper-responsive regulator CsoR is indirectly involved in Bradyrhizobium diazoefficiens denitrification.

The soybean endosymbiont Bradyrhizobium diazoefficiens harbours the complete denitrification pathway that is catalysed by a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a nitrous oxide reductase (Nos), encoded by the napEDABC, nirK, norCBQD, and nosRZDFYLX genes, respectively. Induction of denitrification genes requires low oxygen and nitric oxide, both signals integrated into a complex regulatory network comprised by two interconnected cascades, FixLJ-FixK2-NnrR and RegSR-NifA. Copper is a cofactor of NirK and Nos, but it has also a role in denitrification gene expression and protein synthesis. In fact, Cu limitation triggers a substantial down-regulation of nirK, norCBQD, and nosRZDFYLX gene expression under denitrifying conditions. Bradyrhizobium diazoefficiens genome possesses a gene predicted to encode a Cu-responsive repressor of the CsoR family, which is located adjacent to copA, a gene encoding a putative Cu+-ATPase transporter. To investigate the role of CsoR in the control of denitrification gene expression in response to Cu, a csoR deletion mutant was constructed in this work. Mutation of csoR did not affect the capacity of B. diazoefficiens to grow under denitrifying conditions. However, by using qRT-PCR analyses, we showed that nirK and norCBQD expression was much lower in the csoR mutant compared to wild-type levels under Cu-limiting denitrifying conditions. On the contrary, copA expression was significantly increased in the csoR mutant. The results obtained suggest that CsoR acts as a repressor of copA. Under Cu limitation, CsoR has also an indirect role in the expression of nirK and norCBQD genes.

Bacterial nitric oxide reductase (NorBC) models employing click chemistry.

Bacterial NO Reductase (NorBC or cNOR) is a membrane-bound enzyme found in denitrifying bacteria that catalyzes the two-electron reduction of NO to N2O and water. The mechanism by which NorBC operates is highly debated, due to the fact that this enzyme is difficult to work with, and no intermediates of the NO reduction reaction could have been identified so far. The unique active site of NorBC consists of a heme b3/non-heme FeB diiron center. Synthetic model complexes provide the opportunity to obtain insight into possible mechanistic alternatives for this enzyme. In this paper, we present three new synthetic model systems for NorBC, consisting of a tetraphenylporphyrin-derivative clicked to modified BMPA-based ligands (BMPA = bis(methylpyridyl)amine) that model the non-heme site in the enzyme. These complexes have been characterized by EPR, IR and UV-Vis spectroscopy. The reactivity with NO was then investigated, and it was found that the complex with the BMPA-carboxylate ligand as the non-heme component has a very low affinity for NO at the non-heme iron site. If the carboxylate functional group is replaced with a phenolate or pyridine group, reactivity is restored and formation of a diiron dinitrosyl complex was observed. Upon one-electron reduction of the nitrosylated complexes, following the semireduced pathway for NO reduction, formation of dinitrosyl iron complexes (DNICs) was observed in all three cases, but no N2O could be detected.

Insights into the structure-function relationship of the NorQ/NorD chaperones from Paracoccus denitrificans reveal shared principles of interacting MoxR AAA+/VWA domain proteins.

BACKGROUND: NorQ, a member of the MoxR-class of AAA+ ATPases, and NorD, a protein containing a Von Willebrand Factor Type A (VWA) domain, are essential for non-heme iron (FeB) cofactor insertion into cytochrome c-dependent nitric oxide reductase (cNOR). cNOR catalyzes NO reduction, a key step of bacterial denitrification. This work aimed at elucidating the specific mechanism of NorQD-catalyzed FeB insertion, and the general mechanism of the MoxR/VWA interacting protein families. RESULTS: We show that NorQ-catalyzed ATP hydrolysis, an intact VWA domain in NorD, and specific surface carboxylates on cNOR are all features required for cNOR activation. Supported by BN-PAGE, low-resolution cryo-EM structures of NorQ and the NorQD complex show that NorQ forms a circular hexamer with a monomer of NorD binding both to the side and to the central pore of the NorQ ring. Guided by AlphaFold predictions, we assign the density that "plugs" the NorQ ring pore to the VWA domain of NorD with a protruding "finger" inserting through the pore and suggest this binding mode to be general for MoxR/VWA couples. CONCLUSIONS: Based on our results, we present a tentative model for the mechanism of NorQD-catalyzed cNOR remodeling and suggest many of its features to be applicable to the whole MoxR/VWA family.

Direct electrochemical detection of extracellular nitric oxide in Arabidopsis protoplast based on cytochrome P450 55B1 biosensor.

Cytochrome P450 55B1(CYP55B1) from Chlamydomonas reinhardtii reduces nitric oxide (NO) to dinitrogen oxide (N2O) with the electron supply from NAD(P)H in vivo. Here a novel nitric oxide biosensor was developed by immobilized CYP55B1 on the surface of pyrolytic graphite electrode (PGE) by cross-linking with glutaraldehyde (GA) and bovine serum albumin (BSA). The direct electrochemistry of CYP55B1 was realized with the redox peak potential of -0.355 V and -0.385 V and the catalytic reduction peak of NO by CYP55B1 is at -0.85 V at the scan rate of 0.5 V S-1 in pH 7.0 phosphate buffer. The apparent coverage (Γ = 1.43 × 10-11 mol cm-2), the electron transfer rate constant (ks = 17.39 s-1) and apparent affinity to NO (Kmapp = 11.64 nM) of CYP55B1 in GA/BSA film were obtained. The catalytic mechanism of CYP55B1 towards NO with NADH was examined by the biosensor. The linear range of NO detection was investigated by differential pulse voltammetry with the results of 5-50 nM and the detection limit of 0.5 nM (S/N = 3). The selectivity and stability of the electrochemical biosensor were investigated. Furthermore, the CYP55B1electrochemical biosensor was applied to monitor NO release from Arabidopsis protoplasts with the average content of 0.848 fmol per cell under anaerobic condition.

Effect of Copper on Expression of Functional Genes and Proteins Associated with Bradyrhizobium diazoefficiens Denitrification.

Nitrous oxide (N2O) is a powerful greenhouse gas that contributes to climate change. Denitrification is one of the largest sources of N2O in soils. The soybean endosymbiont Bradyrhizobium diazoefficiens is a model for rhizobial denitrification studies since, in addition to fixing N2, it has the ability to grow anaerobically under free-living conditions by reducing nitrate from the medium through the complete denitrification pathway. This bacterium contains a periplasmic nitrate reductase (Nap), a copper (Cu)-containing nitrite reductase (NirK), a c-type nitric oxide reductase (cNor), and a Cu-dependent nitrous oxide reductase (Nos) encoded by the napEDABC, nirK, norCBQD and nosRZDFYLX genes, respectively. In this work, an integrated study of the role of Cu in B. diazoefficiens denitrification has been performed. A notable reduction in nirK, nor, and nos gene expression observed under Cu limitation was correlated with a significant decrease in NirK, NorC and NosZ protein levels and activities. Meanwhile, nap expression was not affected by Cu, but a remarkable depletion in Nap activity was found, presumably due to an inhibitory effect of nitrite accumulated under Cu-limiting conditions. Interestingly, a post-transcriptional regulation by increasing Nap and NirK activities, as well as NorC and NosZ protein levels, was observed in response to high Cu. Our results demonstrate, for the first time, the role of Cu in transcriptional and post-transcriptional control of B. diazoefficiens denitrification. Thus, this study will contribute by proposing useful strategies for reducing N2O emissions from agricultural soils.

Novel nitrite reductase domain structure suggests a chimeric denitrification repertoire in the phylum Chloroflexi.

Denitrification plays a central role in the global nitrogen cycle, reducing and removing nitrogen from marine and terrestrial ecosystems. The flux of nitrogen species through this pathway has a widespread impact, affecting ecological carrying capacity, agriculture, and climate. Nitrite reductase (Nir) and nitric oxide reductase (NOR) are the two central enzymes in this pathway. Here we present a previously unreported Nir domain architecture in members of phylum Chloroflexi. Phylogenetic analyses of protein domains within Nir indicate that an ancestral horizontal transfer and fusion event produced this chimeric domain architecture. We also identify an expanded genomic diversity of a rarely reported NOR subtype, eNOR. Together, these results suggest a greater diversity of denitrification enzyme arrangements exist than have been previously reported.

Analyzing the mechanism of nitrous oxide production in aerobic phase of anoxic/aerobic sequential batch reactor from the perspective of key enzymes.

How the vast majority of nitrous oxide (N2O) in the aerobic zone of nitrogen bio-removal process is produced is still a controversial issue. To solve this issue, this study measured the activities of two key denitrifying enzymes (nitric oxide reductase (Nor) and nitrous oxide reductase (N2OR)) in an A/O SBR with different chemical nitrogen demand (COD)/total nitrogen (TN) ratios. By analyzing the Spearman's correlations between the N2O production, the enzyme activities, and the factors, the main N2O production process was identified. By comparing the activities of these enzymes, this study analyzed the reasons for the N2O production. Results show that Nor activities had a linear relationship with total N2O concentrations (y = 0.34749 + 31.31365x, R2 = 0.83362) and were not affected by COD (r = 0.299, N = 15, P = 0.279 > 0.05), which showed that most of the N2O released and produced came from the autotrophic denitrification. N2OR activities had a positive correlation with COD (r = 0.692, N = 15, P = 0.004 < 0.01), which showed that heterotrophic denitrification played a role as an N2O consumer. Nor activities were much higher than N2OR activities and the gap between them increased when the total N2O concentration increased, showing that the heterotrophic denitrification was difficult to consume all the N2O produced by the autotrophic denitrification. Reducing autotrophic denitrification is the best way to reduce N2O production in aerobic phase.

Distribution of Denitrification among Haloarchaea: A Comprehensive Study.

Microorganisms from the Halobacteria class, also known as haloarchaea, inhabit a wide range of ecosystems of which the main characteristic is the presence of high salt concentration. These environments together with their microbial communities are not well characterized, but some of the common features that they share are high sun radiation and low availability of oxygen. To overcome these stressful conditions, and more particularly to deal with oxygen limitation, some microorganisms drive alternative respiratory pathways such as denitrification. In this paper, denitrification in haloarchaea has been studied from a phylogenetic point of view. It has been demonstrated that the presence of denitrification enzymes is a quite common characteristic in Halobacteria class, being nitrite reductase and nitric oxide reductase the enzymes with higher co-occurrence, maybe due to their possible role not only in denitrification, but also in detoxification. Moreover, copper-nitrite reductase (NirK) is the only class of respiratory nitrite reductase detected in these microorganisms up to date. The distribution of this alternative respiratory pathway and their enzymes among the families of haloarchaea has also been discussed and related with the environment in which they constitute the major populations. Complete denitrification phenotype is more common in some families like Haloarculaceae and Haloferacaceae, whilst less common in families such as Natrialbaceae and Halorubraceae.

Bacterial nitric oxide metabolism: Recent insights in rhizobia.

Nitric oxide (NO) is a reactive gaseous molecule that has several functions in biological systems depending on its concentration. At low concentrations, NO acts as a signaling molecule, while at high concentrations, it becomes very toxic due to its ability to react with multiple cellular targets. Soil bacteria, commonly known as rhizobia, have the capacity to establish a N2-fixing symbiosis with legumes inducing the formation of nodules in their roots. Several reports have shown NO production in the nodules where this gas acts either as a signaling molecule which regulates gene expression, or as a potent inhibitor of nitrogenase and other plant and bacteria enzymes. A better understanding of the sinks and sources of NO in rhizobia is essential to protect symbiotic nitrogen fixation from nitrosative stress. In nodules, both the plant and the microsymbiont contribute to the production of NO. From the bacterial perspective, the main source of NO reported in rhizobia is the denitrification pathway that varies significantly depending on the species. In addition to denitrification, nitrate assimilation is emerging as a new source of NO in rhizobia. To control NO accumulation in the nodules, in addition to plant haemoglobins, bacteroids also contribute to NO detoxification through the expression of a NorBC-type nitric oxide reductase as well as rhizobial haemoglobins. In the present review, updated knowledge about the NO metabolism in legume-associated endosymbiotic bacteria is summarized.

N2O and NOy production by the comammox bacterium Nitrospira inopinata in comparison with canonical ammonia oxidizers.

Nitrous oxide (N2O) and NOy (nitrous acid (HONO) + nitric oxide (NO) + nitrogen dioxide (NO2)) are released as byproducts or obligate intermediates during aerobic ammonia oxidation, and further influence global warming and atmospheric chemistry. The ammonia oxidation process is catalyzed by groups of globally distributed ammonia-oxidizing microorganisms, which are playing a major role in atmospheric N2O and NOy emissions. Yet, little is known about HONO and NO2 production by the recently discovered, widely distributed complete ammonia oxidizers (comammox), able to individually perform the oxidation of ammonia to nitrate via nitrite. Here, we examined the N2O and NOy production patterns by comammox bacterium Nitrospira inopinata during aerobic ammonia oxidation, in comparison to its canonical ammonia-converting counterparts, representatives of the ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). Our findings, i) show low yield NOy production by the comammox bacterium compared to AOB; ii) highlight the role of the NO reductase in the biological formation of N2O based on results from NH2OH inhibition assays and its stimulation during archaeal and bacterial ammonia oxidations; iii) postulate that the lack of hydroxylamine (NH2OH) and NO transformation enzymatic activities may lead to a buildup of NH2OH/NO which can abiotically react to N2O ; iv) collectively confirm restrained N2O and NOy emission by comammox bacteria, an unneglectable consortium of microbes in global atmospheric emission of reactive nitrogen gases.

The active form of quinol-dependent nitric oxide reductase from Neisseria meningitidis is a dimer.

Neisseria meningitidis is carried by nearly a billion humans, causing developmental impairment and over 100 000 deaths a year. A quinol-dependent nitric oxide reductase (qNOR) plays a critical role in the survival of the bacterium in the human host. X-ray crystallographic analyses of qNOR, including that from N. meningitidis (NmqNOR) reported here at 3.15 Šresolution, show monomeric assemblies, despite the more active dimeric sample being used for crystallization. Cryo-electron microscopic analysis of the same chromatographic fraction of NmqNOR, however, revealed a dimeric assembly at 3.06 Šresolution. It is shown that zinc (which is used in crystallization) binding near the dimer-stabilizing TMII region contributes to the disruption of the dimer. A similar destabilization is observed in the monomeric (∼85 kDa) cryo-EM structure of a mutant (Glu494Ala) qNOR from the opportunistic pathogen Alcaligenes (Achromobacter) xylosoxidans, which primarily migrates as a monomer. The monomer-dimer transition of qNORs seen in the cryo-EM and crystallographic structures has wider implications for structural studies of multimeric membrane proteins. X-ray crystallographic and cryo-EM structural analyses have been performed on the same chromatographic fraction of NmqNOR to high resolution. This represents one of the first examples in which the two approaches have been used to reveal a monomeric assembly in crystallo and a dimeric assembly in vitrified cryo-EM grids. A number of factors have been identified that may trigger the destabilization of helices that are necessary to preserve the integrity of the dimer. These include zinc binding near the entry of the putative proton-transfer channel and the preservation of the conformational integrity of the active site. The mutation near the active site results in disruption of the active site, causing an additional destabilization of helices (TMIX and TMX) that flank the proton-transfer channel helices, creating an inert monomeric enzyme.

Heterologous expression and functional study of nitric oxide reductase catalytic reduction peptide from Achromobacter denitrificans strain TB.

Biological denitrification is a promising and green technology for air pollution control. To investigate the nitric oxide reductase (NOR) that dominates NO reduction efficiency in biological purification, the heterologous prokaryotic expression system of the norB gene, which encodes the core peptide of the catalytic reduction structure in the NOR from Achromobacter denitrificans strain TB, was constructed in Escherichia coli BL21 (DE3). Results showed that the 1218 bp-long norB gene was expressed at the highest level under 1.0 mM IPTG for 5 h at 30 °C, and the relative expression abundance of norB in recombinant E. coli was increased by 16.6 times compared with that of the wild-type TB. However, the NO reduction efficiency and NOR activity of strain TB was 2.7 and 1.83 times higher than those of recombinant E. coli, respectively. On the basis of genomic reassembly and protein structure modeling, the core peptide of the NOR catalytic reduction structure from Achromobacter sp. TB can independently exert NO reduction. The low NO degradation efficiency of recombinant E. coli may be due to the lack of a NorC-like structure that increases the enzyme activity of the NorB protein. The results of this study can be used as basis for further research on the structure and function of NOR.

Quantifying Nitric Oxide Flux Distributions.

Nitric oxide (NO) is a radical that is used as an attack molecule by immune cells. NO can interact and damage a range of biomolecules, and the biological outcome for bacteria assaulted with NO will be governed by how the radical distributes within their biochemical reaction networks. Measurement of those NO fluxes is complicated by the low abundance and transience of many of its reaction products. To overcome this challenge, we use computational modeling to translate measurements of several biochemical species (e.g., NO, O2, NO2-) into NO flux distributions. In this chapter, we provide a detailed protocol, which includes experimental measurements and computational modeling, to estimate the NO flux distribution in an Escherichia coli culture. Those fluxes will have uncertainty associated with them and we also discuss how further experiments and modeling can be employed for flux refinement.

The Response of nor and nos Contributes to Staphylococcus aureus Virulence and Metabolism.

Staphylococcus aureus causes a wide spectrum of disease, with the site and severity of infection dependent on virulence traits encoded within genetically distinct clonal complexes (CCs) and bacterial responses to host innate immunity. The production of nitric oxide (NO) by activated phagocytes is a major host response to which S. aureus metabolically adapts through multiple strategies that are conserved in all CCs, including an S. aureus nitric oxide synthase (Nos). Previous genome analysis of CC30, a lineage associated with chronic endocardial and osteoarticular infections, revealed a putative NO reductase (Nor) not found in other CCs that potentially contributes to NO resistance and clinical outcome. Here, we demonstrate that Nor has true nitric oxide reductase activity, with nor expression enhanced by NO stress and anaerobic growth. Furthermore, we demonstrate that nor is regulated by MgrA and SrrAB, which modulate S. aureus virulence and hypoxic response. Transcriptome analysis of the S. aureus UAMS-1, UAMS-1 Δnor, and UAMS-1 Δnos strains under NO stress and anaerobic growth demonstrates that Nor contributes to nucleotide metabolism and Nos to glycolysis. We demonstrate that Nor and Nos contribute to enhanced survival in the presence of human human polymorphonuclear cells and have organ-specific seeding in a tail vein infection model. Nor contributes to abscess formation in an osteological implant model. We also demonstrate that Nor has a role in S. aureus metabolism and virulence. The regulation overlap between Nor and Nos points to an intriguing link between regulation of intracellular NO, metabolic adaptation, and persistence in the CC30 lineage.IMPORTANCEStaphylococcus aureus can cause disease at most body sites, and illness spans asymptomatic infection to death. The variety of clinical presentations is due to the diversity of strains, which are grouped into distinct clonal complexes (CCs) based on genetic differences. The ability of S. aureus CC30 to cause chronic infections relies on its ability to evade the oxidative/nitrosative defenses of the immune system and survive under different environmental conditions, including differences in oxygen and nitric oxide concentrations. The significance of this work is the exploration of unique genes involved in resisting NO stress and anoxia. A better understanding of the functions that control the response of S. aureus CC30 to NO and oxygen will guide the treatment of severe disease presentations.

Biosensor for direct bioelectrocatalysis detection of nitric oxide using nitric oxide reductase incorporated in carboxylated single-walled carbon nanotubes/lipidic 3 bilayer nanocomposite.

An enzymatic biosensor based on nitric oxide reductase (NOR; purified from Marinobacter hydrocarbonoclasticus) was developed for nitric oxide (NO) detection. The biosensor was prepared by deposition onto a pyrolytic graphite electrode (PGE) of a nanocomposite constituted by carboxylated single-walled carbon nanotubes (SWCNTs), a lipidic bilayer [1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-di-(9Z-octadecenoyl)-3-trimethylammonium-propane (DOTAP), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG)] and NOR. NOR direct electron transfer and NO bioelectrocatalysis were characterized by several electrochemical techniques. The biosensor development was also followed by scanning electron microscopy and Fourier transform infrared spectroscopy. Improved enzyme stability and electron transfer (1.96 × 10-4 cm.s-1 apparent rate constant) was obtained with the optimum SWCNTs/(DOPE:DOTAP:DSPE-PEG)/NOR) ratio of 4/2.5/4 (v/v/v), which biomimicked the NOR environment. The PGE/[SWCNTs/(DOPE:DOTAP:DSPE-PEG)/NOR] biosensor exhibited a low Michaelis-Menten constant (4.3 µM), wide linear range (0.44-9.09 µM), low detection limit (0.13 µM), high repeatability (4.1% RSD), reproducibility (7.0% RSD), and stability (ca. 5 weeks). Selectivity tests towards L-arginine, ascorbic acid, sodium nitrate, sodium nitrite and glucose showed that these compounds did not significantly interfere in NO biosensing (91.0 ±â€¯9.3%-98.4 ±â€¯5.3% recoveries). The proposed biosensor, by incorporating the benefits of biomimetic features of the phospholipid bilayer with SWCNT's inherent properties and NOR bioelectrocatalytic activity and selectivity, is a promising tool for NO.

Electroanalytical characterization of the direct Marinobacter hydrocarbonoclasticus nitric oxide reductase-catalysed nitric oxide and dioxygen reduction.

Understanding the direct electron transfer processes between redox proteins and electrode surface is fundamental to understand the proteins mechanistic properties and for development of novel biosensors. In this study, nitric oxide reductase (NOR) extracted from Marinobacter hydrocarbonoclasticus bacteria was adsorbed onto a pyrolytic graphite electrode (PGE) to develop an unmediated enzymatic biosensor (PGE/NOR)) for characterization of NOR direct electrochemical behaviour and NOR electroanalytical features towards NO and O2. Square-wave voltammetry showed the reduction potential of all the four NOR redox centers: 0.095 ±â€¯0.002, -0.108 ±â€¯0.008, -0.328 ±â€¯0.001 and -0.635 ±â€¯0.004 V vs. SCE for heme c, heme b, heme b3 and non-heme FeB, respectively. The determined sensitivity (-4.00 × 10-8 ±â€¯1.84 × 10-9 A/µM and - 2.71 × 10-8 ±â€¯1.44 × 10-9 A/µM for NO and O2, respectively), limit of detection (0.5 µM for NO and 1.0 µM for O2) and the Michaelis Menten constant (2.1 and 7.0 µM for NO and O2, respectively) corroborated the higher affinity of NOR for its natural substrate (NO). No significant interference on sensitivity towards NO was perceived in the presence of O2, while the O2 reduction was markedly and negatively impacted (3.6 times lower sensitivity) by the presence of NO. These results clearly demonstrate the high potential of NOR for the design of innovative NO biosensors.

Heme redox potentials hold the key to reactivity differences between nitric oxide reductase and heme-copper oxidase.

Despite high structural homology between NO reductases (NORs) and heme-copper oxidases (HCOs), factors governing their reaction specificity remain to be understood. Using a myoglobin-based model of NOR (FeBMb) and tuning its heme redox potentials (E°') to cover the native NOR range, through manipulating hydrogen bonding to the proximal histidine ligand and replacing heme b with monoformyl (MF-) or diformyl (DF-) hemes, we herein demonstrate that the E°' holds the key to reactivity differences between NOR and HCO. Detailed electrochemical, kinetic, and vibrational spectroscopic studies, in tandem with density functional theory calculations, demonstrate a strong influence of heme E°' on NO reduction. Decreasing E°' from +148 to -130 mV significantly impacts electronic properties of the NOR mimics, resulting in 180- and 633-fold enhancements in NO association and heme-nitrosyl decay rates, respectively. Our results indicate that NORs exhibit finely tuned E°' that maximizes their enzymatic efficiency and helps achieve a balance between opposite factors: fast NO binding and decay of dinitrosyl species facilitated by low E°' and fast electron transfer facilitated by high E°'. Only when E°' is optimally tuned in FeBMb(MF-heme) for NO binding, heme-nitrosyl decay, and electron transfer does the protein achieve multiple (>35) turnovers, previously not achieved by synthetic or enzyme-based NOR models. This also explains a long-standing question in bioenergetics of selective cross-reactivity in HCOs. Only HCOs with heme E°' in a similar range as NORs (between -59 and 200 mV) exhibit NOR reactivity. Thus, our work demonstrates efficient tuning of E°' in various metalloproteins for their optimal functionality.