A novel conformer of oxidized Paracoccus pantotrophus cytochrome cd(1) observed by freeze-quench NIR-MCD spectroscopy

Paracoccus pantotrophus cytochrome cd(1) is a physiological nitrite reductase and an in vitro hydroxylamine reductase. The oxidised "as isolated" form of the enzyme has bis-histidinyl coordinated c-heme and upon reduction its coordination changes to histidine/methionine. Following treatment of reduced enzyme with hydroxylamine, a novel, oxidised, conformer of the enzyme is obtained. We have devised protocols for freeze-quench near-ir-MCD spectroscopy that have allowed us to establish unequivocally the c-heme coordination of this species as His/Met. Thus it is shown that the catalytically competent, hydroxylamine reoxidised, form of P. pantotrophus cytochrome cd(1) has different axial ligands to the c-heme than "as isolated" enzyme.     

Pseudoazurin dramatically enhances the reaction profile of nitrite reduction by Paracoccus pantotrophus cytochrome cd1 and facilitates release of product nitric oxide

Cytochrome cd(1) is a respiratory nitrite reductase found in the periplasm of denitrifying bacteria. When fully reduced Paracoccus pantotrophus cytochrome cd(1) is mixed with nitrite in a stopped-flow apparatus in the absence of excess reductant, a kinetically stable complex of enzyme and product forms, assigned as a mixture of cFe(II) d(1)Fe(II)-NO(+) and cFe(III) d(1)Fe(II)-NO (cd(1)-X). However, in order for the enzyme to achieve steady-state turnover, product (NO) release must occur. In this work, we have investigated the effect of a physiological electron donor to cytochrome cd(1), the copper protein pseudoazurin, on the mechanism of nitrite reduction by the enzyme. Our data clearly show that initially oxidized pseudoazurin causes rapid further turnover by the enzyme to give a final product that we assign as all-ferric cytochrome cd(1) with nitrite bound to the d(1) heme (i.e. from which NO had dissociated). Pseudoazurin catalyzed this effect even when present at only one-tenth the stoichiometry of cytochrome cd(1). In contrast, redox-inert zinc pseudoazurin did not affect cd(1)-X, indicating a crucial role for electron movement between monomers or individual enzyme dimers rather than simply a protein-protein interaction. Furthermore, formation of cd(1)-X was, remarkably, accelerated by the presence of pseudoazurin, such that it occurred at a rate consistent with cd(1)-X being an intermediate in the catalytic cycle. It is clear that cytochrome cd(1) functions significantly differently in the presence of its two substrates, nitrite and electron donor protein, than in the presence of nitrite alone.     

Activation of the cytochrome cd1 nitrite reductase from Paracoccus pantotrophus. Reaction of oxidized enzyme with substrate drives a ligand switch at heme c

Cytochromes cd(1) are dimeric bacterial nitrite reductases, which contain two hemes per monomer. On reduction of both hemes, the distal ligand of heme d(1) dissociates, creating a vacant coordination site accessible to substrate. Heme c, which transfers electrons from donor proteins into the active site, has histidine/methionine ligands except in the oxidized enzyme from Paracoccus pantotrophus where both ligands are histidine. During reduction of this enzyme, Tyr(25) dissociates from the distal side of heme d(1), and one heme c ligand is replaced by methionine. Activity is associated with histidine/methionine coordination at heme c, and it is believed that P. pantotrophus cytochrome cd(1) is unreactive toward substrate without reductive activation. However, we report here that the oxidized enzyme will react with nitrite to yield a novel species in which heme d(1) is EPR-silent. Magnetic circular dichroism studies indicate that heme d(1) is low-spin Fe(III) but EPR-silent as a result of spin coupling to a radical species formed during the reaction with nitrite. This reaction drives the switch to histidine/methionine ligation at Fe(III) heme c. Thus the enzyme is activated by exposure to its physiological substrate without the necessity of passing through the reduced state. This reactivity toward nitrite is also observed for oxidized cytochrome cd(1) from Pseudomonas stutzeri suggesting a more general involvement of the EPR-silent Fe(III) heme d(1) species in nitrite reduction.     

Cytochrome cd1, reductive activation and kinetic analysis of a multifunctional respiratory enzyme.

Paracoccus pantotrophus cytochrome cd(1) is an enzyme of bacterial respiration, capable of using nitrite in vivo and also hydroxylamine and oxygen in vitro as electron acceptors. We present a comprehensive analysis of the steady state kinetic properties of the enzyme with each electron acceptor and three electron donors, pseudoazurin and cytochrome c(550), both physiological, and the non-physiological horse heart cytochrome c. At pH 5.8, optimal for nitrite reduction, the enzyme has a turnover number up to 121 s(-1) per d(1) heme, significantly higher than previously observed for any cytochrome cd(1). Pre-activation of the enzyme via reduction is necessary to establish full catalytic competence with any of the electron donor proteins. There is no significant kinetic distinction between the alternative physiological electron donors in any respect, providing support for the concept of pseudospecificity, in which proteins with substantially different tertiary structures can transfer electrons to the same acceptor. A low level hydroxylamine disproportionase activity that may be an intrinsic property of cytochromes c is also reported. Important implications for the enzymology of P. pantotrophus cytochrome cd(1) are discussed and proposals are made about the mechanism of reduction of nitrite, based on new observations placed in the context of recent rapid reaction studies.     

The Structure of an alternative form of Paracoccus pantotrophus cytochrome cd(1) nitrite reductase

Cytochrome cd(1) nitrite reductase is a bifunctional enzyme, which can catalyze the 1-electron reduction of nitrite to nitric oxide and the 4-electron reduction of dioxygen to water. Here we describe the structure of reduced nitrite reductase, crystallized under anaerobic conditions. The structure reveals substantial domain rearrangements with the c domain rotated by 60 degrees and shifted by approximately 20 A compared with previously known structures from crystals grown under oxidizing conditions. This alternative conformation gives rise to different electron transfer routes between the c and d(1) domains and points to the involvement of elements of very large structural changes in the function in this enzyme. In the present structure, the c heme has a His-69/Met-106 ligation, and this ligation does not change upon oxidation in the crystal. The d(1) heme is penta-coordinated, and the d(1) iron is displaced from the heme plane by 0.5 A toward the proximal ligand, His-200. After oxidation, the iron moves into the d(1) heme plane. A surprising finding is that although reduced nitrite reductase can be readily oxidized by dioxygen in the new crystal, it cannot turnover with its other substrate, nitrite. The results suggest that the rearrangement of the domains affects catalysis and substrate selectivity.     

Heme ligation and conformational plasticity in the isolated c domain of cytochrome cd1 nitrite reductase

The heme ligation in the isolated c domain of Paracoccus pantotrophus cytochrome cd(1) nitrite reductase has been characterized in both oxidation states in solution by NMR spectroscopy. In the reduced form, the heme ligands are His69-Met106, and the tertiary structure around the c heme is similar to that found in reduced crystals of intact cytochrome cd1 nitrite reductase. In the oxidized state, however, the structure of the isolated c domain is different from the structure seen in oxidized crystals of intact cytochrome cd1, where the c heme ligands are His69-His17. An equilibrium mixture of heme ligands is present in isolated oxidized c domain. Two-dimensional exchange NMR spectroscopy shows that the dominant species has His69-Met106 ligation, similar to reduced c domains. This form is in equilibrium with a high-spin form in which Met106 has left the heme iron. Melting studies show that the midpoint of unfolding of the isolated c domain is 320.9 +/- 1.2 K in the oxidized and 357.7 +/- 0.6 K in the reduced form. The thermally denatured forms are high-spin in both oxidation states. The results reveal how redox changes modulate conformational plasticity around the c heme and show the first key steps in the mechanism that lead to ligand switching in the holoenzyme. This process is not solely a function of the properties of the c domain. The role of the d1 heme in guiding His17 to the c heme in the oxidized holoenzyme is discussed.     

Mechanism of spinach chloroplast ferredoxin-dependent nitrite reductase: spectroscopic evidence for intermediate states

Nitrite reductases found in plants, algae, and cyanobacteria catalyze the six-electron reduction of nitrite to ammonia with reduced ferredoxin serving as the electron donor. They contain one siroheme and one [4Fe-4S] cluster, acting as separate one-electron carriers. Nitrite is thought to bind to the siroheme and to remain bound until its complete reduction to ammonia. In the present work the enzyme catalytic cycle, with ferredoxin reduced by photosystem 1 as an electron donor, has been studied by EPR and laser flash absorption spectroscopy. Substrate depletion during enzyme turnover, driven by a series of laser flashes, has been demonstrated. A complex of ferrous siroheme with NO, formed by two-electron reduction of the enzyme complex with nitrite, has been shown to be an intermediate in the enzyme catalytic cycle. The same complex can be formed by incubation of free oxidized nitrite reductase with an excess of nitrite and ascorbate. Hydroxylamine, another putative intermediate in the reduction of nitrite catalyzed by nitrite reductase, was found to react with oxidized nitrite reductase to produce the same ferrous siroheme-NO complex, with a characteristic formation time of about 13 min. The rate-limiting step for this reaction is probably hydroxylamine binding to the enzyme, with the conversion of hydroxylamine to NO at the enzyme active site likely being much faster.     

Oxidase reaction of cytochrome cd(1) from Paracoccus pantotrophus

Cytochrome cd(1) (cd(1)NIR) from Paracoccus pantotrophus, which is both a nitrite reductase and an oxidase, was reduced by ascorbate plus hexaamineruthenium(III) chloride on a relatively slow time scale (hours required for complete reduction). Visible absorption spectroscopy showed that mixing of ascorbate-reduced enzyme with oxygen at pH = 6.0 resulted in the rapid oxidation of both types of heme center in the enzyme with a linear dependence on oxygen concentration. Subsequent changes on a longer time scale reflected the formation and decay of partially reduced oxygen species bound to the d(1) heme iron. Parallel freeze-quench experiments allowed the X-band electron paramagnetic resonance (EPR) spectrum of the enzyme to be recorded at various times after mixing with oxygen. On the same millisecond time scale that simultaneous oxidation of both heme centers was seen in the optical experiments, two new EPR signals were observed. Both of these are assigned to oxidized heme c and resemble signals from the cytochrome c domain of a "semi-apo" form of the enzyme for which histidine/methionine coordination was demonstrated spectroscopically. These observations suggests that structural changes take around the heme c center that lead to either histidine/methionine axial ligation or a different stereochemistry of bis-histidine axial ligation than that found in the as prepared enzyme. At this stage in the reaction no EPR signal could be ascribed to Fe(III) d(1) heme. Rather, a radical species, which is tentatively assigned to an amino acid radical proximal to the d(1) heme iron in the Fe(IV)-oxo state, was seen. The kinetics of decay of this radical species match the generation of a new form of the Fe(III) d(1) heme, probably representing an OH(-)-bound species. This sequence of events is interpreted in terms of a concerted two-electron reduction of oxygen to bound peroxide, which is immediately cleaved to yield water and an Fe(IV)-oxo species plus the radical. Two electrons from ascorbate are subsequently transferred to the d(1) heme active site via heme c to reduce both the radical and the Fe(IV)-oxo species to Fe(III)-OH(-) for completion of a catalytic cycle.     

The partial characterization of purified nitrite reductase and hydroxylamine oxidase from Nitrosomonas europaea

Nitrite reductase has been separated from cell-free extracts of Nitrosomonas and partially purified from hydroxylamine oxidase by polyacrylamide-gel electrophoresis. In its oxidized state the enzyme, which did not contain haem, had an extinction maximum at 590nm, which was abolished on reduction. Sodium diethyldithiocarbamate was a potent inhibitor of nitrite reductase. Enzyme activity was stimulated 2.5-fold when remixed with hydroxylamine oxidase, but was unaffected by mammalian cytochrome c. The enzyme also exhibited a low hydroxylamine-dependent nitrite reductase activity. The results suggest that this enzyme is similar to the copper-containing ;denitrifying enzyme' of Pseudomonas denitrificans. A dithionite-reduced, 465nm-absorbing haemoprotein was associated with homogeneous preparations of hydroxylamine oxidase. The band at 465nm maximum was not reduced during the oxidation of hydroxylamine although the extinction was abolished on addition of hydroxylamine, NO(2) (-) or CO. These last-named compounds when added to the oxidized enzyme precluded the appearance of the 465nm-absorption band on addition of dithionite. Several properties of 465nm-absorbing haemoprotein are described.     

Reactions of spinach nitrite reductase with its substrate, nitrite, and a putative intermediate, hydroxylamine

Plant nitrite reductase (NiR) catalyzes the reduction of nitrite (NO(2)(-)) to ammonia, using reduced ferredoxin as the electron donor. NiR contains a [4Fe-4S] cluster and an Fe-siroheme, which is the nitrite binding site. In the enzyme's as-isolated form ([4Fe-4S](2+)/Fe(3+)), resonance Raman spectroscopy indicated that the siroheme is in the high-spin ferric hexacoordinated state with a weak sixth axial ligand. Kinetic and spectroscopic experiments showed that the reaction of NiR with NO(2)(-) results in an unexpectedly EPR-silent complex formed in a single step with a rate constant of 0.45 +/- 0.01 s(-)(1). This binding rate is slow compared to that expected from the NiR turnover rates reported in the literature, suggesting that binding of NO(2)(-) to the as-isolated form of NiR is not the predominant type of substrate binding during enzyme turnover. Resonance Raman spectroscopic characterization of this complex indicated that (i) the siroheme iron is low-spin hexacoordinated ferric, (ii) the ligand coordination is unusually heterogeneous, and (iii) the ligand is not nitric oxide, most likely NO(2)(-). The reaction of oxidized NiR with hydroxylamine (NH(2)OH), a putative intermediate, results in a ferrous siroheme-NO complex that is spectroscopically identical to the one observed during NiR turnover. Resonance Raman and absorption spectroscopy data show that the reaction of oxidized NiR ([4Fe-4S](2+)/Fe(3+)) with hydroxylamine is binding-limited, while the NH(2)OH conversion to nitric oxide is much faster.     

Protein film voltammetry reveals distinctive fingerprints of nitrite and hydroxylamine reduction by a cytochrome C nitrite reductase

The cytochrome c nitrite reductases perform a key step in the biological nitrogen cycle by catalyzing the six-electron reduction of nitrite to ammonium. Graphite electrodes painted with Escherichia coli cytochrome c nitrite reductase and placed in solutions containing nitrite (pH 7) exhibit large catalytic reduction currents during cyclic voltammetry at potentials below 0 V. These catalytic currents were not observed in the absence of cytochrome c nitrite reductase and were shown to originate from an enzyme film engaged in direct electron exchange with the electrode. The catalytic current-potential profiles observed on progression from substrate-limited to enzyme-limited nitrite reduction revealed a fingerprint of catalytic behavior distinct from that observed during hydroxylamine reduction, the latter being an alternative substrate for the enzyme that is reduced to ammonium in a two electron process. Cytochrome c nitrite reductase clearly interacts differently with these two substrates. However, similar features underlie the development of the voltammetric response with increasing nitrite or hydroxylamine concentration. These features are consistent with coordinated two-electron reduction of the active site and suggest that the mechanisms for reduction of both substrates are underpinned by common rate-defining processes.     

An iron dioxygenase from Alcaligenes faecalis catalyzing the oxidation of pyruvic oxime to nitrite

Abstract An enzyme which participated in the oxidation of hydroxylamine to nitrite from was partially purified Alcaligenes faecalis , and some of its properties were studied. The enzyme oxidized aerobically pyruvic oxime to nitrite in the presence of hydroxylamine or ascorbate. As molecular oxygen equimolar to nitrite formed was consumed in the enzymatic oxidation of pyruvic oxime to nitrite, the enzyme was thought to be a dioxygenase. It was an iron protein, and a reducing reagent was required to keep the iron in the ferrous state for the action of the enzyme.     

Electron paramagnetic resonance studies of heme c and its nitrosyl derivative in Vibrio (Achromobacter) fischeri nitrite reductase

Interactions of Vibrio (formerly Achromobacter) fischeri nitrite reductase were studied by electron paramagnetic resonance spectroscopy. The spectrum of the oxidized enzyme showed a number of features which were attributed to two low-spin ferric hemes. These comprised an unusual derivative peak at g = 3.7 and a spectrum at g = 2.88, 2.26, and 1.51. Neither heme was reactive in the oxidized state with the substrate nitrite and with cyanide and azide. When frozen under turnover conditions (i.e., reduction in the presence of excess nitrite), the enzyme showed the spectrum of a nitrosyl heme derivative. The g = 2.88, 2.26, and 1.51 signals reappeared partially on reoxidation by nitrite, indicating that the nitrosyl species which remained arose from the g = 3.7 heme. The nitrosyl derivative showed a 14N nuclear hyperfine splitting, Az = 1.65 mT. The nitrosyl derivative was produced by treatment of the oxidized nitrite reductase with nitric oxide or hydroxylamine. Exchange of nitric oxide between the nitrosyl derivative and NO gas in solution was observed by using the [15N]nitrosyl compound. A possible reaction cycle for the enzyme is discussed, which involves reduction of the enzyme followed by binding of nitrite to one heme and formation of the nitrosyl intermediate.     

A cytochrome cd1-type nitrite reductase isolated from the marine denitrifier Pseudomonas nautica 617: purification and characterization

Nitrite reductase (cytochrome cd1) was purified to electrophoretic homogeneity from the soluble extract of the marine denitrifying bacterium Pseudomonas nautica strain 617. Cells were anaerobically grown with 10 mM nitrate as final electron acceptor. The soluble fraction was purified by four successive chromatographic steps and the purest cytochrome cd1 exhibited an A280 nm(oxidized)/A410nm(oxidized) coefficient of 0.90. In the course of purification, cytochrome cd1 specific activity presented a maximum value of 0.048 units/mg of protein. This periplasmic enzyme is a homodimer and each 60 kDa subunit contains one heme c and one heme d1 as prosthetic moieties, both in a low spin state. Redox potentials of hemes c and d1 were determined at three different pH values (6.6, 7.6 and 8.6) and did not show any pH dependence. The first 20 amino acids of the NH2-terminal region of the protein were identified and the sequence showed 45% identity with the corresponding region of Pseudomonas aeruginosa nitrite reductase but no homology to Pseudomonas stutzeri and Paracoccus denitrificans enzymes. Spectroscopic properties of Pseudomonas nautica 617 cytochrome cd1 in the ultraviolet-visible range and in electron paramagnetic resonance are described. The formation of a heme d1 -nitric-oxide complex as an intermediate of nitrite reduction was demonstrated by electron paramagnetic resonance experiments.     

Y25S variant of Paracoccus pantotrophus cytochrome cd1 provides insight into anion binding by d1 heme and a rare example of a critical difference between solution and crystal structures

Tyr25 is a ligand to the active site d1 heme in as isolated, oxidized cytochrome cd1 nitrite reductase from Paracoccus pantotrophus. This form of the enzyme requires reductive activation, a process that involves not only displacement of Tyr25 from the d1 heme but also switching of the ligands at the c heme from bis-histidinyl to His/Met. A Y25S variant retains this bis-histidinyl coordination in the crystal of the oxidized state that has sulfate bound to the d1 heme iron. This Y25S form of the enzyme does not require reductive activation, an observation previously interpreted as meaning that the presence of the phenolate oxygen of Tyr25 is the critical determinant of the requirement for activation. This interpretation now needs re-evaluation because, unexpectedly, the oxidized as prepared Y25S protein, unlike the wild type, has different heme iron ligands in solution at room temperature, as judged by magnetic circular dichroism and electron spin resonance spectroscopies, than in the crystal. In addition, the binding of nitrite and cyanide to oxidized Y25S cytochrome cd1 is markedly different from the wild type enzyme, thus providing insight into the affinity of the oxidized d1 heme ring for anions in the absence of the steric barrier presented by Tyr25.     

Oxidation of hydroxylamine to nitrite catalyzed by hydroxylamine oxidoreductase purified fromNitrosomonas europaea

The oxidation of hydroxylamine to nitrite, which had been catalyzed by hydroxylamine oxidoreductase purified fromNitrosomonas europaea, was studied. The enzyme oxidized hydroxylamine almost completely to nitrite under aerobic conditions if sufficient amount of cytochromec or ferricyanide was added and the reaction was performed in phosphate buffer. Even under anaerobic conditions, hydroxylamine was oxidized to nitrite by the enzyme, but nitrite, once formed, disappeared when the reaction was continued for more than several minutes.     

Identification of the sources of nitrous oxide produced by oxidative and reductive processes in Nitrosomonas europaea

1. Cells of Nitrosomonas europaea produced N(2)O during the oxidation of ammonia and hydroxylamine. 2. The end-product of ammonia oxidation, nitrite, was the predominant source of N(2)O in cells. 3. Cells also produced N(2)O, but not N(2) gas, by the reduction of nitrite under anaerobic conditions. 4. Hydroxylamine was oxidized by cell-free extracts to yield nitrite and N(2)O aerobically, but to yield N(2)O and NO anaerobically. 5. Cell extracts reduced nitrite both aerobically and anaerobically to NO and N(2)O with hydroxylamine as an electron donor. 6. The relative amounts of NO and N(2)O produced during hydroxylamine oxidation and/or nitrite reduction are dependent on the type of artificial electron acceptor utilized. 7. Partially purified hydroxylamine oxidase retained nitrite reductase activity but cytochrome oxidase was absent. 8. There is a close association of hydroxylamine oxidase and nitrite reductase activities in purified preparations.     

Nitrite and nitric oxide treatment of Helix pomatia hemocyanin: single and double oxidation of the active site.

The reaction of nitrite and nitric oxide with Helix pomatia hemocyanin has been studied. One or both of the two copper ions in the active site can be oxidized, depending upon reaction conditions. The single oxidation of the oxygen binding site can be reversed by reduction with hydroxylamine, and the oxygen binding properties of the protein are simultaneously restored. The experiments, including electron paramagnetic resonance, indicate that nitric oxide is not a ligand of copper in the singly oxidized active site and that the oxidized copper ions is coupled to at least two nitrogen atoms of amino acid residues. The doubly oxidized protein can be reduced to a singly oxidized one with ascorbic acid or hydroxylamine; the latter reagent is again able to reduce the singly oxidized state and to restore the oxygen binding properties.     

Nitrite reductase of Escherichia coli specific for reduced nicotinamide adenine dinucleotide

Kemp, John D. (University of California, Los Angeles), and Daniel E. Atkinson. Nitrite reductase of Escherichia coli specific for reduced nicotinamide adenine dinucleotide. J. Bacteriol. 92:628-634. 1966.-A nitrite reductase specific for reduced nicotinamide adenine dinucleotide (NADH(2)) appears to be responsible for in vivo nitrite reduction by Escherichia coli strain Bn. In extracts, the reduction product is ammonium, and the ratio of NADH(2) oxidized to nitrite reduced or to ammonium produced is 3. The Michaelis constant for nitrite is 10 mum. The enzyme is induced by nitrite, and the ability of intact cells to reduce nitrite parallels the level of NADH(2)-specific nitrite reductase activity demonstrable in cell-free preparations. Crude extracts of strain Bn will also reduce hydroxylamine, but not nitrate or sulfite, at the expense of NADH(2). Kinetic observations indicate that hydroxylamine and nitrite may both be reduced at the same active site. The high apparent Michaelis constant for hydroxylamine (1.5 mm), however, seems to exclude hydroxylamine as an intermediate in nitrite reduction. In vitro activity is enhanced by preincubation with nitrite, and decreased by preincubation with NADH(2).