Bacterial Nitric Oxide Reductase Research Articles

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.

Cytochrome cM Is Probably a Membrane Protein Similar to the C Subunit of the Bacterial Nitric Oxide Reductase

Cytochrome cM was first described in 1994 and its sequence has been found in the genome of manifold cyanobacterial species ever since. Numerous studies have been carried out with the purpose of determining its function, but none of them has given place to conclusive results so far. Many of these studies are based on the assumption that cytochrome cM is a soluble protein located in the thylakoid lumen of cyanobacteria. In this work, we have reevaluated the sequence of cytochrome cM, with our results showing that its most probable 3D structure is strongly similar to that of the C subunit of the bacterial nitric oxide reductase. The potential presence of an α-helix tail, which could locate this protein in the thylakoid membrane, further supports this hypothesis, thus providing a new, unexpected role for this redox protein.

Timing of NO Binding and Protonation in the Catalytic Reaction of Bacterial Nitric Oxide Reductase as Established by Time-Resolved Spectroscopy

Abstract Membrane-integrated nitric oxide reductases (NOR) catalyze the formation of nitrous oxide (N2O) from two NO molecules using two protons and two electrons at a heme/non-heme iron binuclear center. Despite extensive efforts, the mechanism underlying the NOR-catalyzed reaction has been poorly understood due to the rapidity of the reaction. Here, we utilized a photosensitive caged NO compound as a trigger for the NOR reaction to characterize the NO reduction mechanism by time-resolved visible absorption spectroscopy. We showed that the NOR reaction consists of three steps. One NO molecule binds to the reduced binuclear center to form a non-heme Fe(II)-NO species in the 1st phase (microsecond timescale), followed by a migration of NO to form the other chemical species, possibly 5-coordinate heme b3-NO, in the 2nd phase (timescale of tens of microseconds). Then, the NO bound to heme reacts with a second NO molecule in the 3rd phase (millisecond timescale), in which protonation and electron transfer promote N-N bond formation and N-O bond cleavage to yield N2O. These findings led us to propose a revised trans mechanism for NO reduction by NOR.

Nitric Oxide Reductase Activity in Heme-Nonheme Binuclear Engineered Myoglobins through a One-Electron Reduction Cycle.

FeBMbs are structural and functional models of native bacterial nitric oxide reductases (NORs) generated through engineering of myoglobin. These biosynthetic models replicate the heme-nonheme diiron site of NORs and allow substitutions of metal centers and heme cofactors. Here, we provide evidence for multiple NOR turnover in monoformyl-heme-containing FeBMb1 proteins loaded with FeII, CoII, or ZnII metal ions at the FeB site (FeII/CoII/ZnII-FeBMb1(MF-heme)). FTIR detection of the ν(NNO) band of N2O at 2231 cm-1 provides a direct quantitative measurement of the product in solution. A maximum number of turnover is observed with FeII-FeBMb1(MF-heme), but the NOR activity is retained when the FeB site is loaded with ZnII. These data support the viability of a one-electron semireduced pathway for the reduction of NO at binuclear centers in reducing conditions.

Is Endophyte-Plant Co-Denitrification a Source of Nitrous Oxides Emission? —An Experimental Investigation with Soybean

The biological pattern of plants’ nitrous oxide (N2O) generation is not well understood because plant cells cannot form N2O from nitric oxide (NO) reduction. Hypothetically, we consider that plant-endophytic co-denitrification is potentially capable of making up for the functional loss of NO reduction in the plant cell and is thus the source of plant-derived N2O. In order to test the above hypothesis, field-cultured soybean seedlings and aseptic-cultured seedlings were used to establish plant holobionts with differentially established endophytes. The N2O fluxes, copies of the bacterial NO reductase encoding gene, and a diversity of endophytic denitrifying bacteria of these holobionts were observed by gas chromatography, real-time PCR, and 16s rDNA sequencing. The flux of N2O by the field seedlings was significantly higher than the fluxes of the aseptic seedlings and bulk soil. The N2O flux of the soybean seedlings was significantly correlated to the abundance of the bacterial NO reductase encoding gene. Eleven genera of denitrifying bacteria were observed in the soybean seedlings, and among them, two genera of aerobic denitrifying bacteria were specifically associated with colonizing plant samples. In this work we have also showed that plant N2O emission is affected by the abundance of total endophytic denitrifying bacteria. We conclude that plant sourced N2O is cooperatively generated by the plant-endophyte symbiotic system in which endophytic denitrifying bacteria help plants to emit N2O by taking over the NO–N2O reduction process.

Coordination and Electronic Structures of Short-Lived Intermediate of Heme-Containing NO Reductases

Nitric oxide reductases (NOR) are heme-containing enzymes, which are involved in fungal or bacterial denitrification, and reduce nitric oxide (NO) to nitrous oxide (N2O) fro detoxification. Fungal NOR is a water-soluble and cytochrome P450 type enzyme, while bacterial NOR is a membrane-integrated enzyme containing a heme/non-heme iron binuclear center as its active site. In both enyzmes, short-lived intermediates are appeared during the NO reduction reaction. For characterizing the coordination and electronic structures of the intermediates, we applied time-resolved IR spectroscopic and crystallographic techniques. The information obtained from these experiments will help us to understand the molecular mechanism of the NO reduction catalyzed by two different NOR enzymes.

Characterization of the quinol-dependent nitric oxide reductase from the pathogen Neisseria meningitidis, an electrogenic enzyme

Bacterial nitric oxide reductases (NORs) catalyse the reduction of NO to N2O and H2O. NORs are found either in denitrification chains, or in pathogens where their primary role is detoxification of NO produced by the immune defense of the host. Although NORs belong to the heme-copper oxidase superfamily, comprising proton-pumping O2-reducing enzymes, the best studied NORs, cNORs (cytochrome c-dependent), are non-electrogenic. Here, we focus on another type of NOR, qNOR (quinol-dependent). Recombinant qNOR from Neisseria meningitidis, a human pathogen, purified from Escherichia coli, showed high catalytic activity and spectroscopic properties largely similar to cNORs. However, in contrast to cNOR, liposome-reconstituted qNOR showed respiratory control ratios above two, indicating that NO reduction by qNOR was electrogenic. Further, we determined a 4.5 Å crystal structure of the N. meningitidis qNOR, allowing exploration of a potential proton transfer pathway from the cytoplasm by mutagenesis. Most mutations had little effect on the activity, however the E-498 variants were largely inactive, while the corresponding substitution in cNOR was previously shown not to induce significant effects. We thus suggest that, contrary to cNOR, the N. meningitidis qNOR uses cytoplasmic protons for NO reduction. Our results allow possible routes for protons to be discussed.

In Situ Surface Enhanced Infrared Absorption Spectroscopy for Nitric Oxide Reduction Catalyzed By Bacterial Nitric Oxide Reductase

In microbial denitrification process, nitrate (NO3 −), which is the main nitrogen-containing pollutant in ground water, is stepwise reduced to harmless dinitrogen (N2) thorough nitrite (NO2 −), nitric oxide (NO) and nitrous oxide (N2O). Although ideally no intermediate metabolite is lost to the environment, certain soil bacteria release N2O into the atmosphere. N2O is known to contribute to the green house gas effect and to ozone depletion. The largest emission source of N2O is bacterial breakdown of nitrogen compounds and the key enzyme in the N2O production is a metalloenzyme of nitric oxide reductase (NOR). NOR is a membrane-integrated protein and catalyzes the reduction of NO to N2O at active sites of iron co-factors of the non-heme FeB and/or the heme b 3[1].The NO reduction involves the N−O bond cleavage and the N−N formation. The reaction mechanism is interesting from a chemical point of view because the NO reduction is known to govern the selectivity of the final product, N2 or NH3, in electrochemical nitrate reduction[2].Three possible reaction mechanisms are proposed: trans-mechanism, cis-heme b 3 mechanism and cis-FeB mechanism[3].However, the molecular mechanism of the NO reduction catalyzed by NOR still remains to be solved. Herein, we report protein film voltammetry (PFV) and surface enhanced infrared absorption (SEIRA) spectroscopy of NOR at gold electrodes to gain mechanistic insights into the NO reduction. PFV allows us to apply electrochemical potentials to redox-active enzymes including NOR on the electrode surface[4] and to initiate electrocatalytic reactions by the enzyme. SEIRA spectroscopy is a powerful technique to understand reaction mechanisms of enzymes at the electrode surface[5].In this work, a cytochrome c-dependent NOR was isolated from Pseudomonas Aeruginosa and then immobilized directly on the surface of a gold-coated Si hemicylinder prism[6] or via self-assembled monolayers for cyclic voltammetry and SEIRA spectroscopy. Cyclic voltammograms of NOR-modified electrodes showed reduction current under NO but not under Ar, suggesting that NOR on the electrode surface catalyzed the NO reduction to N2O. Potential-dependent SEIRA spectra of NOR-modified electrodes were recorded under NO and characteristic two bands were observed in the range from 1600 cm−1 to 1800 cm−1. NO molecules bonded to the non-heme FeB and to the heme b 3are known to show absorption bands in this IR range[6].Thus, it is most likely that NOR on the electrode may catalyze the NO reduction in the trans mechanism.

(Invited) Molecular Mechanism of NO Reduction By Bacterial Nitric Oxide Reductases

Bacterial nitric oxide reductase (NOR) catalyzes the NO reduction reaction (2NO + 2H+ + 2e- --> N2O + H2O) at its active site, which is a binuclear center comprised by heme (heme b 3) and non-heme iron (FeB). In the enzymatic reaction by NOR, three short-lived intermediates have been proposed for the coordination of two molecules of NO to the binuclear center; i.e., trans-, cis-heme b 3- and cis-FeB-coordinations. To establish the molecular mechanism of the NO reduction by NOR, we have been studying the time-resolved spectroscopies to characterize the coordination and electronic structures of the intermediates, which are appeared in the micro- to milli-second time region after the reaction of the ferrous enzyme with the NO molecule(s). On the basis of the time-resolved optical, ESR and IR spectral changes, we have proposed that the trans-mechanism seem favorable for the NO reduction mechanism by bacterial NOR. We will discuss the NOR reaction with referring their crystallographic structural data which we reported.

A bridged di-iron porphyrin hyponitrite complex as a model for biological N2O production from hyponitrite

Heme-hyponitrites are intermediates that form at the bimetallic active sites of bacterial nitric oxide reductases. To probe a possible effect of the Fe–Fe distance on hyponitrite stability, we prepared a bridged bis-porphyrin Fe-hyponitrite compound, namely [(OEP-CH2)Fe]2(μ2,η1,η1-ONNO). Its υNO of 992 cm−1 (υ15NO of 976 cm−1) is close to the υNO of 983 cm−1 reported previously by us for the crystallographically characterized [(OEP)Fe]2(μ2,η1,η1-ONNO) compound. The bridged bis-porphyrin Fe-hyponitrite complex is unstable with respect to N2O production, supporting the role of the bis-Fe porphyrin system in hyponitrite conversion to N2O. The preparation and crystallographic determination of the bridging sulfato derivative is also reported.

Bacterial nitric oxide reductase: a mechanism revisited by an ONIOM (DFT:MM) study.

Bacterial nitric oxide reductase (cNOR) is an important binuclear iron enzyme responsible for the reduction of nitric oxide to nitrous oxide in the catalytic cycle of bacterial respiration. The reaction mechanism of cNOR as well as the key reactive intermediates of the reaction are still under debate. Here, we report a computational study based on ONIOM (DFT:MM) calculations aimed at investigating the reaction mechanism of cNOR. The results suggest that the reaction proceeds via the mono-nitrosyl mechanism which starts off by the binding of an NO molecule to the heme b3 center, N-N hyponitrite bond formation as a result of the reaction with a second NO molecule was found to proceed with an exothermic energy barrier to yield a hyponitrite adduct forming an open (incomplete) ring conformation with the non-heme FeB center (O-N-N-O-FeB). N-O bond cleavage to yield N2O was shown to be the rate-limiting step with an activation barrier of 22.6kcal mol(-1). The dinitrosyl (trans) mechanism, previously proposed by several studies, was also examined and found unfavorable due to high activation barriers of the resulting intermediates.

Characterization of the bridged hyponitrite complex {[Fe(OEP)](2)(μ-N(2)O(2))}: reactivity of hyponitrite complexes and biological relevance.

The detoxification of nitric oxide (NO) by bacterial NO reductase (NorBC) represents a paradigm of how NO can be detoxified anaerobically in cells. In order to elucidate the mechanism of this enzyme, model complexes provide a convenient means to assess potential reaction intermediates. In particular, there have been many proposed mechanisms that invoke the formation of a hyponitrite bridge between the heme b3 and nonheme iron (FeB) centers within the NorBC active site. However, the reactivity of bridged iron hyponitrite complexes has not been investigated much in the literature. The model complex {[Fe(OEP)]2(μ-N2O2)} offers a unique opportunity to study the electronic structure and reactivity of such a hyponitrite-bridged complex. Here we report the detailed characterization of {[Fe(OEP)]2(μ-N2O2)} using a combination of IR, nuclear resonance vibrational spectroscopy, electron paramagnetic resonance, and magnetic circular dichroism spectroscopy along with SQUID magnetometry. These results show that the ground-state electronic structure of this complex is best described as having two intermediate-spin (S = (3)/2) iron centers that are weakly antiferromagnetically coupled across the N2O2(2-) bridge. The analogous complex {[Fe(PPDME)]2(μ-N2O2)} shows overall similar properties. Finally, we report the unexpected reaction of {[Fe(OEP)]2(μ-N2O2)} in the presence and absence of 1-methylimidizole to yield [Fe(OEP)(NO)]. Density functional theory calculations are used to rationalize why {[Fe(OEP)]2(μ-N2O2)} cannot be formed directly by dimerization of [Fe(OEP)(NO)] and why only the reverse reaction is observed experimentally. These results thus provide insight into the general reactivity of hyponitrite-bridged iron complexes with general relevance for the N-N bond-forming step in NorBC.

3P092 一酸化窒素還元酵素における基質NO結合の分子機構(02. ヘム蛋白質,ポスター,第52回日本生物物理学会年会(2014年度))

3P092 一酸化窒素還元酵素における基質NO結合の分子機構(02. ヘム蛋白質,ポスター,第52回日本生物物理学会年会(2014年度))

Unexpected weak magnetic exchange coupling between haem and non-haem iron in the catalytic site of nitric oxide reductase (NorBC) from Paracoccus denitrificans

Bacterial NOR (nitric oxide reductase) is a major source of the powerful greenhouse gas N2O. NorBC from Paracoccus denitrificans is a heterodimeric multi-haem transmembrane complex. The active site, in NorB, comprises high-spin haem b3 in close proximity with non-haem iron, FeB. In oxidized NorBC, the active site is EPR-silent owing to exchange coupling between FeIII haem b3 and FeBIII (both S=5/2). On the basis of resonance Raman studies [Moënne-Loccoz, Richter, Huang, Wasser, Ghiladi, Karlin and de Vries (2000) J. Am. Chem. Soc. 122, 9344-9345], it has been assumed that the coupling is mediated by an oxo-bridge and subsequent studies have been interpreted on the basis of this model. In the present study we report a VFVT (variable-field variable-temperature) MCD (magnetic circular dichroism) study that determines an isotropic value of J=-1.7 cm-1 for the coupling. This is two orders of magnitude smaller than that encountered for oxo-bridged diferric systems, thus ruling out this configuration. Instead, it is proposed that weak coupling is mediated by a conserved glutamate residue.

一酸化窒素還元酵素の結晶構造と呼吸酵素の分子進化

脱窒は，嫌気性微生物の呼吸の一種であり，硝酸イオンNO3-を順次還元し，最終的に窒素N2に変換する生物プロセスである．その変換過程において中間体として生成される一酸化窒素NOは，ラジカル分子であり，タンパク質などの生体物質に損傷を与えるため，微生物は，NO生成後速やかに無毒化する一酸化窒素還元酵素NORを有している．この膜結合性NORの結晶構造が世界で初めて解かれた．これにより，微生物によるNO無毒化の反応機構を明らかにし，呼吸酵素の分子進化を解明できる手がかりが得られた．

Electrochemical behaviour of bacterial nitric oxide reductase—Evidence of low redox potential non-heme FeB gives new perspectives on the catalytic mechanism

Nitric oxide reductase (NOR) is a membrane bound enzyme involved in the metabolic denitrification pathway, reducing nitric oxide (NO) to nitrous oxide (N2O), subsequently promoting the formation of the NN bond. Three types of bacterial NOR are known, namely cNOR, qNOR and qCuNOR, that differ on the physiological electron donor. cNOR has been purified as a two subunit complex, the NorC, anchored to the cytoplasmic membrane, with a low-spin heme c, and the NorB subunit showing high structural homology with the HCuO subunit I, comprising a bis-histidine low-spin heme b and a binuclear iron centre. The binuclear iron centre is the catalytic site and it is formed by a heme b3 coupled to a non-heme iron (FeB) through a μ-oxo bridge. The catalytic mechanism is still under discussion and three hypotheses have been proposed: the trans-mechanism, the cis-FeB and the cis-heme b3 mechanisms. In the present work, the Pseudomonas nautica cNOR electrochemical behaviour was studied by cyclic voltammetry (CV), using a pyrolytic graphite electrode modified with the immobilised protein. The protein redox centres were observed and the formal redox potentials were determined. The binuclear iron centre presents the lowest redox potential value, and discrimination between the heme b3 and FeB redox processes was attained. Also, the number of electrons involved and correspondent surface electronic transfer rate constants were estimated. The pH dependence of the observed redox processes was determined and some new insights on the NOR catalytic mechanism are discussed.

Molecular Dynamics Simulations Reveal Proton Transfer Pathways in Cytochrome C-Dependent Nitric Oxide Reductase

Nitric oxide reductases (NORs) are membrane proteins that catalyze the reduction of nitric oxide (NO) to nitrous oxide (N2O), which is a critical step of the nitrate respiration process in denitrifying bacteria. Using the recently determined first crystal structure of the cytochrome c-dependent NOR (cNOR) [Hino T, Matsumoto Y, Nagano S, Sugimoto H, Fukumori Y, et al. (2010) Structural basis of biological N2O generation by bacterial nitric oxide reductase. Science 330: 1666–70.], we performed extensive all-atom molecular dynamics (MD) simulations of cNOR within an explicit membrane/solvent environment to fully characterize water distribution and dynamics as well as hydrogen-bonded networks inside the protein, yielding the atomic details of functionally important proton channels. Simulations reveal two possible proton transfer pathways leading from the periplasm to the active site, while no pathways from the cytoplasmic side were found, consistently with the experimental observations that cNOR is not a proton pump. One of the pathways, which was newly identified in the MD simulation, is blocked in the crystal structure and requires small structural rearrangements to allow for water channel formation. That pathway is equivalent to the functional periplasmic cavity postulated in cbb 3 oxidase, which illustrates that the two enzymes share some elements of the proton transfer mechanisms and confirms a close evolutionary relation between NORs and C-type oxidases. Several mechanisms of the critical proton transfer steps near the catalytic center are proposed.

Structural basis for NO reduction by bacterial nitric oxide reductases

Structural basis for NO reduction by bacterial nitric oxide reductases

Structural basis for nitrous oxide generation by bacterial nitric oxide reductases

The crystal structure of the bacterial nitric oxide reductase (cNOR) from Pseudomonas aeruginosa is reported. Its overall structure is similar to those of the main subunit of aerobic and micro-aerobic cytochrome oxidases (COXs), in agreement with the hypothesis that all these enzymes are members of the haem-copper oxidase superfamily. However, substantial structural differences between cNOR and COX are observed in the catalytic centre and the delivery pathway of the catalytic protons, which should be reflected in functional differences between these respiratory enzymes. On the basis of the cNOR structure, we propose a possible reaction mechanism of nitric oxide reduction to nitrous oxide as a working hypothesis.

Proton transfer in the quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus during reduction of oxygen

Bacterial nitric oxide reductases (NOR) are integral membrane proteins that catalyse the reduction of nitric oxide to nitrous oxide, often as a step in the process of denitrification. Most functional data has been obtained with NORs that receive their electrons from a soluble cytochrome c in the periplasm and are hence termed cNOR. Very recently, the structure of a different type of NOR, the quinol-dependent (q)-NOR from the thermophilic bacterium Geobacillus stearothermophilus was solved to atomic resolution [Y. Matsumoto, T. Tosha, A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat. Struct. Mol. Biol. 19 (2012) 238–246]. In this study, we have investigated the reaction between this qNOR and oxygen. Our results show that, like some cNORs, the G. stearothermophilus qNOR is capable of O2 reduction with a turnover of ~3electronss−1 at 40°C. Furthermore, using the so-called flow-flash technique, we show that the fully reduced (with three available electrons) qNOR reacts with oxygen in a reaction with a time constant of 1.8ms that oxidises the low-spin heme b. This reaction is coupled to proton uptake from solution and presumably forms a ferryl intermediate at the active site. The pH dependence of the reaction is markedly different from a corresponding reaction in cNOR from Paracoccus denitrificans, indicating that possibly the proton uptake mechanism and/or pathway differs between qNOR and cNOR. This study furthermore forms the basis for investigation of the proton transfer pathway in qNOR using both variants with putative proton transfer elements modified and measurements of the vectorial nature of the proton transfer. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).