Reduction of nitric oxide in bacterial nitric oxide reductase—a theoretical model study

The mechanism of the nitric oxide reduction in a bacterial nitric oxide reductase (NOR) has been investigated in two model systems of the heme-b₃-Fe_B active site using density functional theory (B3LYP). A model with an octahedral coordination of the non-heme Fe_B consisting of three histidines, one glutamate and one water molecule gave an energetically feasible reaction mechanism. A tetrahedral coordination of the non-heme iron, corresponding to the one of Cu_B in cytochrome oxidase, gave several very high barriers which makes this type of coordination unlikely. The first nitric oxide coordinates to heme b₃ and is partly reduced to a more nitroxyl anion character, which activates it toward an attack from the second NO. The product in this reaction step is a hyponitrite dianion coordinating in between the two irons. Cleaving an NO bond in this intermediate forms an Fe_B (IV)O and nitrous oxide, and this is the rate determining step in the reaction mechanism. In the model with an octahedral coordination of Fe_B the intrinsic barrier of this step is 16.3 kcal/mol, which is in good agreement with the experimental value of 15.9 kcal/mol. However, the total barrier is 21.3 kcal/mol, mainly due to the endergonic reduction of heme b₃ taken from experimental reduction potentials. After nitrous oxide has left the active site the ferrylic Fe_B will form a μ-oxo bridge to heme b₃ in a reaction step exergonic by 45.3 kcal/mol. The formation of a quite stable μ-oxo bridge between heme b₃ and Fe_B is in agreement with this intermediate being the experimentally observed resting state in oxidized NOR. The formation of a ferrylic non-heme Fe_B in the proposed reaction mechanism could be one reason for having an iron as the non-heme metal ion in NOR instead of a Cu as in cytochrome oxidase.     

Diverse NO reduction by Halomonas halodenitrificans nitric oxide reductase

Reduction of the four Fe centers is not required to initiate the reaction of the Halomonas halodenitrificans nitric oxide reductase (NOR) based on the facts that NOR in the form that ferric heme b(3) and non-heme iron (Fe(B)) are not bridged and/or the interaction between them is weakened and reversibly binds NO molecules, and that NOR in the form that only heme b(3) is oxidized reacts with NO molecules.     

A low-redox potential heme in the dinuclear center of bacterial nitric oxide reductase: implications for the evolution of energy-conserving heme-copper oxidases.

Bacterial nitric oxide reductase (NOR) catalyzes the two-electron reduction of nitric oxide to nitrous oxide. It is a highly diverged member of the superfamily of heme-copper oxidases. The main feature by which NOR is distinguished from the heme-copper oxidases is the elemental composition of the active site, a dinuclear center comprised of heme b(3) and non-heme iron (Fe(B)). The visible region electronic absorption spectrum of reduced NOR exhibits a maximum at 551 nm with a distinct shoulder at 560 nm; these are attributed to Fe(II) heme c (E(m) = 310 mV) and Fe(II) heme b (E(m) = 345 mV), respectively. The electronic absorption spectrum of oxidized NOR exhibits a characteristic shoulder around 595 nm that exhibits complex behavior in equilibrium redox titrations. The first phase of reduction is characterized by an apparent shift of the shoulder to 604 nm and a decrease in intensity. This is due to reduction of Fe(B) (E(m) = 320 mV), while the subsequent bleaching of the 604 nm band represents reduction of heme b(3) (E(m) = 60 mV). This separation of redox potentials (>200 mV) allows the enzyme to be poised in the three-electron reduced state for detailed spectroscopic examination of the Fe(III) heme b(3) center. The low midpoint potential of heme b(3) represents a thermodynamic barrier to the complete (two-electron) reduction of the dinuclear center. This may avoid formation of a stable Fe(II) heme b(3)-NO species during turnover, which may be an inhibited state of the enzyme. It would also appear that the evolution of significant oxygen reducing activity by heme-copper oxidases was not simply a matter of the substitution of copper for non-heme iron in the dinuclear center. Changes in the protein environment that modulate the midpoint redox potential of heme b(3) to facilitate both complete reduction of the dinuclear center (a prerequisite for oxygen binding) and rapid heme-heme electron transfer were also necessary.     

Reaction of carbon monoxide with the reduced active site of bacterial nitric oxide reductase.

Bacterial nitric oxide reductase (NOR), a member of the superfamily of heme-copper oxidases, catalyzes the two-electron reduction of nitric oxide to nitrous oxide. The key feature that distinguishes NOR from the typical heme-copper oxidases is the elemental composition of the dinuclear center, which contains non-heme iron (FeB) rather than copper (CuB). UV-vis electronic absorption and room-temperature magnetic circular dichroism (RT-MCD) spectroscopies showed that CO binds to Fe(II) heme b3 to yield a low-spin six-coordinate species. Photolysis of the Fe(II)-CO bond is followed by CO recombination (k(on) = 1.7 x 10(8) M(-1) x s(-1)) that is approximately 3 orders of magnitude faster than CO recombination to the active site of typical heme-copper oxidases (k(on) = 7 x 10(4) M(-1)x s(-1)). This rapid rate of CO recombination suggests an unimpeded pathway to the active site that may account for the enzyme's high affinity for substrate, essential for maintaining denitrification at low concentrations of NO. In contrast, the initial binding of CO to reduced heme b3 measured by stopped-flow spectroscopy is much slower (k(on) = 1.2 x 10(5) M(-1) x s(-1)). This suggests that an existing heme distal ligand (water/OH-) may be displaced to elicit the spin-state change observed in the RT-MCD spectrum.     

A theoretical study on nitric oxide reductase activity in a ba(3)-type heme-copper oxidase

The mechanism of nitric oxide reduction in a ba(3)-type heme-copper oxidase has been investigated using density functional theory (B3LYP). Four possible mechanisms have been studied and free energy surfaces for the whole catalytic cycle including proton and electron transfers have been constructed by comparison to experimental data. The first nitric oxide coordinates to heme a(3) and is partly reduced having some nitroxyl anion character ((3)NO(-)), and it is thus activated toward the attack by the second N-O. In this reaction step a cyclic hyponitrous acid anhydride intermediate with the two oxygens coordinating to Cu(B) is formed. The cyclic hyponitrous acid anhydride is quite stable in a local minimum with high barriers for both the backward and forward reactions and should thus be observable experimentally. To break the N-O bond and form nitrous oxide, the hyponitrous acid anhydride must be protonated, the latter appearing to be an endergonic process. The endergonicity of the proton transfer makes the barrier of breaking the N-O bond directly after the protonation too high. It is suggested that an electron should enter the catalytic cycle at this stage in order to break the N-O bond and form N(2)O at a feasible rate. The cleavage of the N-O bond is the rate limiting step in the reaction mechanism and it has a barrier of 17.3 kcal/mol, close to the experimental value of 19.5 kcal/mol. The overall exergonicity is fitted to experimental data and is 45.6 kcal/mol.     

Molecular structure and function of bacterial nitric oxide reductase

The crystal structure of the membrane-integrated nitric oxide reductase cNOR from Pseudomonas aeruginosa was determined. The smaller NorC subunit of cNOR is comprised of 1 trans-membrane helix and a hydrophilic domain, where the heme c is located, while the larger NorB subunit consists of 12 trans-membrane helices, which contain heme b and the catalytically active binuclear center (heme b(3) and non-heme Fe(B)). The roles of the 5 well-conserved glutamates in NOR are discussed, based on the recently solved structure. Glu211 and Glu280 appear to play an important role in the catalytic reduction of NO at the binuclear center by functioning as a terminal proton donor, while Glu215 probably contributes to the electro-negative environment of the catalytic center. Glu135, a ligand for Ca(2+) sandwiched between two heme propionates from heme b and b(3), and the nearby Glu138 appears to function as a structural factor in maintaining a protein conformation that is suitable for electron-coupled proton transfer from the periplasmic region to the active site. On the basis of these observations, the possible molecular mechanism for the reduction of NO by cNOR is discussed. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Low-spin heme b(3) in the catalytic center of nitric oxide reductase from Pseudomonas nautica

Respiratory nitric oxide reductase (NOR) was purified from membrane extract of Pseudomonas (Ps.) nautica cells to homogeneity as judged by polyacrylamide gel electrophoresis. The purified protein is a heterodimer with subunits of molecular masses of 54 and 18 kDa. The gene encoding both subunits was cloned and sequenced. The amino acid sequence shows strong homology with enzymes of the cNOR class. Iron/heme determinations show that one heme c is present in the small subunit (NORC) and that approximately two heme b and one non-heme iron are associated with the large subunit (NORB), in agreement with the available data for enzymes of the cNOR class. Mo?ssbauer characterization of the as-purified, ascorbate-reduced, and dithionite-reduced enzyme confirms the presence of three heme groups (the catalytic heme b(3) and the electron transfer heme b and heme c) and one redox-active non-heme Fe (Fe(B)). Consistent with results obtained for other cNORs, heme c and heme b in Ps. nautica cNOR were found to be low-spin while Fe(B) was found to be high-spin. Unexpectedly, as opposed to the presumed high-spin state for heme b(3), the Mo?ssbauer data demonstrate unambiguously that heme b(3) is, in fact, low-spin in both ferric and ferrous states, suggesting that heme b(3) is six-coordinated regardless of its oxidation state. EPR spectroscopic measurements of the as-purified enzyme show resonances at the g ～ 6 and g ～ 2-3 regions very similar to those reported previously for other cNORs. The signals at g = 3.60, 2.99, 2.26, and 1.43 are attributed to the two charge-transfer low-spin ferric heme c and heme b. Previously, resonances at the g ～ 6 region were assigned to a small quantity of uncoupled high-spin Fe(III) heme b(3). This assignment is now questionable because heme b(3) is low-spin. On the basis of our spectroscopic data, we argue that the g = 6.34 signal is likely arising from a spin-spin coupled binuclear center comprising the low-spin Fe(III) heme b(3) and the high-spin Fe(B)(III). Activity assays performed under various reducing conditions indicate that heme b(3) has to be reduced for the enzyme to be active. But, from an energetic point of view, the formation of a ferrous heme-NO as an initial reaction intermediate for NO reduction is disfavored because heme [FeNO](7) is a stable product. We suspect that the presence of a sixth ligand in the Fe(II)-heme b(3) may weaken its affinity for NO and thus promotes, in the first catalytic step, binding of NO at the Fe(B)(II) site. The function of heme b(3) would then be to orient the Fe(B)-bound NO molecules for the formation of the N-N bond and to provide reducing equivalents for NO reduction.     

Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen

The respiratory nitric oxide reductase (NOR) from Paracoccus denitrificans catalyzes the two-electron reduction of NO to N(2)O (2NO + 2H(+) + 2e(-) --> N(2)O + H(2)O), which is an obligatory step in the sequential reduction of nitrate to dinitrogen known as denitrification. NOR has four redox-active cofactors, namely, two low-spin hemes c and b, one high-spin heme b(3), and a non-heme iron Fe(B), and belongs to same superfamily as the oxygen-reducing heme-copper oxidases. NOR can also use oxygen as an electron acceptor; this catalytic activity was investigated in this study. We show that the product in the steady-state reduction of oxygen is water. A single turnover of the fully reduced NOR with oxygen was initiated using the flow-flash technique, and the progress of the reaction monitored by time-resolved optical absorption spectroscopy. Two major phases with time constants of 40 micros and 25 ms (pH 7.5, 1 mM O(2)) were observed. The rate constant for the faster process was dependent on the O(2) concentration and is assigned to O(2) binding to heme b(3) at a bimolecular rate constant of 2 x 10(7) M(-)(1) s(-)(1). The second phase (tau = 25 ms) involves oxidation of the low-spin hemes b and c, and is coupled to the uptake of protons from the bulk solution. The rate constant for this phase shows a pH dependence consistent with rate limitation by proton transfer from an internal group with a pK(a) = 6.6. This group is presumably an amino acid residue that is crucial for proton transfer to the catalytic site also during NO reduction.     

Spectral properties of bacterial nitric-oxide reductase: resolution of pH-dependent forms of the active site heme b3

Bacterial nitric-oxide reductase catalyzes the two electron reduction of nitric oxide to nitrous oxide. In the oxidized form the active site non-heme Fe(B) and high spin heme b(3) are mu-oxo bridged. The heme b(3) has a ligand-to-metal charge transfer band centered at 595 nm, which is insensitive to pH over the range of 6.0-8.5. Partial reduction of nitric-oxide reductase yields a three electron-reduced state where only the heme b(3) remains oxidized. This results in a shift of the heme b(3) charge transfer band lambda(max) to longer wavelengths. At pH 6.0 the charge transfer band lambda(max) is 605 nm, whereas at pH 8.5 it is 635 nm. At pH 6.5 and 7.5 the nitric-oxide reductase ferric heme b(3) population is a mixture of both 605- and 635-nm forms. Magnetic circular dichroism spectroscopy suggests that at all pH values examined the proximal ligand to the ferric heme b(3) in the three electron-reduced form is histidine. At pH 8.5 the distal ligand is hydroxide, whereas at pH 6.0, when the enzyme is most active, it is water.     

A theoretical study of myoglobin working as a nitric oxide scavenger

The mechanism for the reaction between nitric oxide (NO) and O₂ bound to the heme iron of myoglobin (Mb), including the following isomerization to nitrate, has been investigated using hybrid density functional theory (B3LYP). Myoglobin working as a NO scavenger could be of importance, since NO reversibly inhibits the terminal enzyme in the respiration chain, cytochrome c oxidase. The concentration of NO in the cell will thus affect the respiration and thereby the synthesis of ATP. The calculations show that the reaction between NO and the heme-bound O₂ gives a peroxynitrite intermediate whose O–O bond undergoes a homolytic cleavage, forming a NO₂ radical and myoglobin in the oxo-ferryl state. The NO₂ radical then recombines with the oxo-ferryl, forming heme-bound nitrate. Nine different models have been used in the present study to examine the effect on the reaction both by the presence and the protonation state of the distal His64, and by the surroundings of the proximal His93. The barriers going from the oxy-Mb and nitric oxide reactant to the peroxynitrite intermediate and further to the oxo-ferryl and NO₂ radical are around 10 and 7 kcal/mol, respectively. Forming the product, nitrate bound to the heme iron has a barrier of less than ~7 kcal/mol. The overall reaction going from a free nitric oxide and oxy-Mb to the heme bound nitrate is exergonic by more than 30 kcal/mol.     

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 O(2) 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).     

Redox-dependent open and closed forms of the active site of the bacterial respiratory nitric-oxide reductase revealed by cyanide binding studies

The bacterial respiratory nitric-oxide reductase (NOR) catalyzes the respiratory detoxification of nitric oxide in bacteria and Archaea. It is a member of the well known super-family of heme-copper oxidases but has a [heme Fe-non-heme Fe] active site rather than the [heme Fe-Cu(B)] active site normally associated with oxygen reduction. Paracoccus denitrificans NOR is spectrally characterized by a ligand-to-metal charge transfer absorption band at 595 nm, which arises from the high spin ferric heme iron of a micro-oxo-bridged [heme Fe(III)-O-Fe(III)] active site. On reduction of the nonheme iron, the micro-oxo bridge is broken, and the ferric heme iron is hydroxylated or hydrated, depending on the pH. At present, the catalytic cycle of NOR is a matter of much debate, and it is not known to which redox state(s) of the enzyme nitric oxide can bind. This study has used cyanide to probe the nature of the active site in a number of different redox states. Our observations suggest that the micro-oxo-bridged [heme Fe(III)-O-Fe(III)] active site represents a closed or resting state of NOR that can be opened by reduction of the non-heme iron.     

Characterization of quinol-dependent nitric oxide reductase from Geobacillus stearothermophilus: Enzymatic activity and active site structure

Nitric oxide reductase (NOR) catalyzes the reduction of nitric oxide to generate nitrous oxide. We recently reported on the crystal structure of a quinol-dependent NOR (qNOR) from Geobacillus stearothermophilus [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], and suggested that a water channel from the cytoplasm, which is not observed in cytochrome c-dependent NOR (cNOR), functions as a pathway transferring catalytic protons. Here, we further investigated the functional and structural properties of qNOR, and compared the findings with those for cNOR. The pH optimum for the enzymatic reaction of qNOR was in the alkaline range, whereas Pseudomonas aeruginosa cNOR showed a higher activity at an acidic pH. The considerably slower reduction rate, and a correlation of the pH dependence for enzymatic activity and the reduction rate suggest that the reduction process is the rate-determining step for the NO reduction by qNOR, while the reduction rate for cNOR was very fast and therefore is unlikely to be the rate-determining step. A close examination of the heme/non-heme iron binuclear center by resonance Raman spectroscopy indicated that qNOR has a more polar environment at the binuclear center compared with cNOR. It is plausible that a water channel enhances the accessibility of the active site to solvent water, creating a more polar environment in qNOR. This structural feature could control certain properties of the active site, such as redox potential, which could explain the different catalytic properties of the two NORs. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Proton and electron pathways in the bacterial nitric oxide reductase

Electron- and proton-transfer reactions in bacterial nitric oxide reductase (NOR) have been investigated by optical spectroscopy and electrometry. In liposomes, NOR does not show any generation of an electric potential during steady-state turnover. This electroneutrality implies that protons are taken up from the same side of the membrane as electrons during catalysis. Intramolecular electron redistribution after photolysis of the partially reduced CO-bound enzyme shows that the electron transfer in NOR has the same pathway as in the heme-copper oxidases. The electron is transferred from the acceptor site, heme c, via a low-spin heme b to the binuclear active site (heme b3/FeB). The electron-transfer rate between hemes c and b is (3 +/- 2) x 10(4) s(-1). The rate of electron transfer between hemes b and b3 is too fast to be resolved (>10(6) s(-1)). Only electron transfer between heme c and heme b is coupled to the generation of an electric potential. This implies that the topology of redox centers in NOR is comparable to that in the heme-copper cytochrome oxidases. The optical and electrometric measurements allow identification of the intermediate states formed during turnover of the fully reduced enzyme, as well as the associated proton and electron movement linked to the NO reduction. The first phase (k = 5 x 10(5) s(-1)) is electrically silent, and characterized by the disappearance of absorbance at 433 nm and the appearance of a broad peak at 410 nm. We assign this phase to the formation of a ferrous NO adduct of heme b3. NO binding is followed by a charge separation phase (k = 2.2 x 10(5) s(-1)). We suggest that the formation of this intermediate that is not linked to significant optical changes involves movement of charged side chains near the active site. The next step creates a negative potential with a rate constant of approximately 3 x 10(4) s(-1) and a weak optical signature. This is followed by an electrically silent phase with a rate constant of 5 x 10(3) s(-1) leading to the last intermediate of the first turnover (a rate constant of approximately 10(3) s(-1)). The fully reduced enzyme has four electrons, enough for two complete catalytic cycles. However, the protons for the second turnover must be taken from the bulk, resulting in the generation of a positive potential in two steps. The optical measurements also verify two phases in the oxidation of low-spin hemes. Based on these results, we present mechanistic models of NO reduction by NOR. The results can be explained with a trans mechanism rather than a cis model involving FeB. Additionally, the data open up the possibility that NOR employs a P450-type mechanism in which only heme b3 functions as the NO binding site during turnover.     

Structures of reduced and ligand‐bound nitric oxide reductase provide insights into functional differences in respiratory enzymes

Nitric oxide reductase (NOR) catalyzes the generation of nitrous oxide (N₂O) via the reductive coupling of two nitric oxide (NO) molecules at a heme/non‐heme Fe center. We report herein on the structures of the reduced and ligand‐bound forms of cytochrome c‐dependent NOR (cNOR) from Pseudomonas aeruginosa at a resolution of 2.3–2.7 Å, to elucidate structure‐function relationships in NOR, and compare them to those of cytochrome c oxidase (CCO) that is evolutionarily related to NOR. Comprehensive crystallographic refinement of the CO‐bound form of cNOR suggested that a total of four atoms can be accommodated at the binuclear center. Consistent with this, binding of bulky acetaldoxime (CH₃‐CH=N‐OH) to the binuclear center of cNOR was confirmed by the structural analysis. Active site reduction and ligand binding in cNOR induced only ～0.5 Å increase in the heme/non‐heme Fe distance, but no significant structural change in the protein. The highly localized structural change is consistent with the lack of proton‐pumping activity in cNOR, because redox‐coupled conformational changes are thought to be crucial for proton pumping in CCO. It also permits the rapid decomposition of cytotoxic NO in denitrification. In addition, the shorter heme/non‐heme Fe distance even in the bulky ligand‐bound form of cNOR (～4.5 Å) than the heme/Cu distance in CCO (～5 Å) suggests the ability of NOR to maintain two NO molecules within a short distance in the confined space of the active site, thereby facilitating N‐N coupling to produce a hyponitrite intermediate for the generation of N₂O. Proteins 2014; 82:1258–1271. © 2013 Wiley Periodicals, Inc.     

NO reduction by nitric-oxide reductase from denitrifying bacterium Pseudomonas aeruginosa: characterization of reaction intermediates that appear in the single turnover cycle

Nitric-oxide reductase (NOR) of a denitrifying bacterium catalyzes NO reduction to N(2)O at the binuclear catalytic center consisting of high spin heme b(3) and non-heme Fe(B). The structures of the reaction intermediates in the single turnover of the NO reduction by NOR from Pseudomonas aeruginosa were investigated using optical absorption and EPR spectroscopies combined with an originally designed freeze-quench device. In the EPR spectrum of the sample, in which the fully reduced NOR was mixed with an NO solution and quenched at 0.5 ms after the mixing, two characteristic signals for the ferrous Fe(B)-NO and the penta-coordinated ferrous heme b(3)-NO species were observed. The CO inhibition of its formation indicated that two NO molecules were simultaneously distributed into the two irons of the same binuclear center of the enzyme in this state. The time- and temperature-dependent EPR spectral changes indicated that the species that appeared at 0.5 ms is a transient reaction intermediate prior to the N(2)O formation, in good agreement with the so-called "trans" mechanism. It was also found that the final state of the enzyme in the single turnover cycle is the fully oxidized state, in which the mu-oxo-bridged ligand is absent between the two irons of its binuclear center, unlike the resting form of NOR as isolated. On the basis of these present findings, we propose a newly developed mechanism for the NO reduction reaction conducted by NOR.     

A theoretical study on nitric oxide reductase activity in a ba3-type heme-copper oxidase

The mechanism of nitric oxide reduction in a ba₃-type heme-copper oxidase has been investigated using density functional theory (B3LYP). Four possible mechanisms have been studied and free energy surfaces for the whole catalytic cycle including proton and electron transfers have been constructed by comparison to experimental data. The first nitric oxide coordinates to heme a₃ and is partly reduced having some nitroxyl anion character (³NO⁻), and it is thus activated toward the attack by the second NO. In this reaction step a cyclic hyponitrous acid anhydride intermediate with the two oxygens coordinating to Cu_B is formed. The cyclic hyponitrous acid anhydride is quite stable in a local minimum with high barriers for both the backward and forward reactions and should thus be observable experimentally. To break the NO bond and form nitrous oxide, the hyponitrous acid anhydride must be protonated, the latter appearing to be an endergonic process. The endergonicity of the proton transfer makes the barrier of breaking the NO bond directly after the protonation too high. It is suggested that an electron should enter the catalytic cycle at this stage in order to break the NO bond and form N₂O at a feasible rate. The cleavage of the NO bond is the rate limiting step in the reaction mechanism and it has a barrier of 17.3 kcal/mol, close to the experimental value of 19.5 kcal/mol. The overall exergonicity is fitted to experimental data and is 45.6 kcal/mol.     

Pseudomonas aeruginosa overexpression system of nitric oxide reductase for in vivo and in vitro mutational analyses

Membrane-integrated nitric oxide reductase (NOR) reduces nitric oxide (NO) to nitrous oxide (N₂O) with protons and electrons. This process is essential for the elimination of the cytotoxic NO that is produced from nitrite (NO₂^?) during microbial denitrification. A structure-guided mutagenesis of NOR is required to elucidate the mechanism for NOR-catalyzed NO reduction. We have already solved the crystal structure of cytochrome c-dependent NOR (cNOR) from Pseudomonas aeruginosa. In this study, we then constructed its expression system using cNOR-gene deficient and wild-type strains for further functional study. Characterizing the variants of the five conserved Glu residues located around the heme/non-heme iron active center allowed us to establish how the anaerobic growth rate of cNOR-deficient strains expressing cNOR variants correlates with the in vitro enzymatic activity of the variants. Since bacterial strains require active cNOR to eliminate cytotoxic NO and to survive under denitrification conditions, the anaerobic growth rate of a strain with a cNOR variant is a good indicator of NO decomposition capability of the variants and a marker for the screening of functionally important residues without protein purification. Using this in vivo screening system, we examined the residues lining the putative proton transfer pathways for NO reduction in cNOR, and found that the catalytic protons are likely transferred through the Glu57 located at the periplasmic protein surface. The homologous cNOR expression system developed here is an invaluable tool for facile identification of crucial residues in vivo, and for further in vitro functional and structural studies.     

Perturbations at the high spin heme b center in the membrane-bound nitric oxide reductase

The effects of lowering pH from 7 to 5 on the absorption, circular dichroism (MCD) and EPR spectra were studied for Paracoccus halodenitrificans nitric oxide reductase (NOR). Intensities of the characteristic bands for the high spin heme b, that at 592 nm in the absorption spectrum and those at 591 (+) and 606 (-) in the MCD spectrum decreased considerably. Concomitant cryogenic EPR spectrum indicated a drastic increase in the signal intensity due to the high spin heme b at g approximately 6, of which less than 5% had been EPR detectable at pH 7. Cyanide (x40) bound to the high spin heme b center in the reduced NOR irrespective of pH, while a much larger amount of azide (x1000) was necessary to bind to the reduced NOR at an acidic pH, ca. 5. Based on these results the structure and function of the high spin heme b center as the active site of NOR was discussed.