Reaction of cytochrome c with nitrite and nitric oxide. A model of dissimilatory nitrite reductase.

The reaction of bovine heart ferrocytochrome c with nitrite was studied under various conditions. The reaction product was ferricytochrome c at around pH 5, whereas at around pH 3 it was Compound I, characterized by twin peaks at 529 and 563 nm of equal intensity. However, ferrocytochrome c decreased obeying first-order kinetics over the pH range examined, irrespective of the presence or absence of molecular oxygen. The apparent first-order rate constant was proportional to the square of the nitrite concentration at pH 4.4 and it increased as the pH was lowered. At pH 3 the reaction was so rapid that it had to be followed by stopped-flow and rapid-scanning techniques. The apparent rate constant at this pH was found to increase linearly with the nitrite concentration. Based on these results the active species of nitrite was concluded to be dinitrogen trioxide at pH 4.4 and nitrosonium ion, no+, at pH 3. Compound II was formed by reaction of ferrocytochrome c and NO gas at acidic and alkaline pH values. The absorption peaks were at 533 and 563 nm at pH 3, and at 538 and 567 nm at pH 12.9. This compound was also formed by reducing Compound I with reductants. Compound I prepared from ferricytochrome c and NO was stable below pH 6. However, appreciable absorption peaks for ferrocytochrome c appeared between pH 8 and 10, because Compound I was dissociated into ferrocytochrome c and NO+, and because ferrocytochrome c thus formed reacted with NO very slowly in this pH region. Saccharomyces ferricytochrome c under NO gas behaved differently from mammalian cytochrome, indicating the significance of the nature of the heme environment in determing the reactivity. Only at extreme pH values was Compound II formed exclusively and persisted. A model system for dissimilatory nitrite reductase was constructed by using bovine heart cytochrome c, nitrite and NADH plus PMS at pH 3.3, and a scheme involving cyclic turnover of ferrocytochrome c, Compound I and Compound II is presented, with kinetic parameters.     

Characterization of a novel copper-haem c dissimilatory nitrite reductase from Ralstonia pickettii

NiRs (nitrite reductases) convert nitrite into NO in the denitrification process. RpNiR (Ralstonia pickettii NiR), a new type of dissimilatory Cu-containing NiR with a C-terminal haem c domain from R. pickettii, has been cloned, overexpressed in Escherichia coli and purified to homogeneity. The enzyme has a subunit molecular mass of 50515 Da, consistent with sequence data showing homology to the well-studied two-domain Cu NiRs, but with an attached C-terminal haem c domain. Gel filtration and combined SEC (size-exclusion chromatography)-SAXS (small angle X-ray scattering) analysis shows the protein to be trimeric. The metal content of RpNiR is consistent with each monomer having a single haem c group and the two Cu sites being metallated by Cu(2+) ions. The absorption spectrum of the oxidized as-isolated recombinant enzyme is dominated by the haem c. X-band EPR spectra have clear features arising from both type 1 Cu and type 2 Cu centres in addition to those of low-spin ferric haem. The requirements for activity and low apparent K(m) for nitrite are similar to other CuNiRs (Cu-centre NiRs). However, EPR and direct binding measurements of nitrite show that oxidized RpNiR binds nitrite very weakly, suggesting that substrate binds to the reduced type 2 Cu site during turnover. Analysis of SEC-SAXS data suggests that the haem c domains in RpNiR form extensions into the solvent, conferring a high degree of conformational flexibility in solution. SAXS data yield R(g) (gyration radius) and D(max) (maximum particle diameter) values of 43.4 ? (1 ?=0.1 nm) and 154 ? compared with 28 ? and 80 ? found for the two-domain CuNiR of Alcaligenes xylosoxidans.     

Heme c - heme d1 interaction in Pseudomonas cytochrome oxidase (nitrite reductase): a reappraisal of the spectroscopic evidence.

Magnetic circular dichroism (MCD) spectra of Pseudomonas aeruginosa cytochrome oxidase are reported over the spectral range of 350–700 nm for the oxidized, ascorbate-reduced, dithionite-reduced and reduced carbon monoxide forms. The spectra of all forms examined can be interpreted as the simple sum of the individual heme c and heme d₁ contributions without invoking “heme-heme interaction.” In particular and contrary to a recent report [Orii, Shimada, Nozawa, and Hatano, this Journal $\text{76}$, 983 (1977)] no effect of ligand binding to ferrous heme d₁ was observed in the MCD spectrum of the heme c component. It seems likely that the previous findings were the result of incomplete reduction of the enzyme in the absence of stabilizing ligands.     

Binding and reduction of sulfite by cytochrome c nitrite reductase

Pentaheme cytochrome c nitrite reductase (ccNiR) catalyzes the six-electron reduction of nitrite to ammonia as the final step in the dissimilatory pathway of nitrate ammonification. It has also been shown to reduce sulfite to sulfide, thus forming the only known link between the biogeochemical cycles of nitrogen and of sulfur. We have found the sulfite reductase activity of ccNiR from Wolinella succinogenes to be significantly smaller than its nitrite reductase activity but still several times higher than the one described for dissimilatory, siroheme-containing sulfite reductases. To compare the sulfite reductase activity of ccNiR with our previous data on nitrite reduction, we determined the binding mode of sulfite to the catalytic heme center of ccNiR from W. succinogenes at a resolution of 1.7 A. Sulfite and nitrite both provide a pair of electrons to form the coordinative bond to the Fe(III) active site of the enzyme, and the oxygen atoms of sulfite are found to interact with the three active site protein residues conserved within the enzyme family. Furthermore, we have characterized the active site variant Y218F of ccNiR that exhibited an almost complete loss of nitrite reductase activity, while sulfite reduction remained unaffected. These data provide a first direct insight into the role of the first sphere of protein ligands at the active site in ccNiR catalysis.     

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.     

Intramolecular electron transfer in cytochrome cd(1) nitrite reductase from Pseudomonas stutzeri; kinetics and thermodynamics

Cytochrome cd(1) nitrite reductase from Pseudomonas stutzeri catalyzes the one electron reduction of nitrite to nitric oxide. It is a homodimer, each monomer containing one heme-c and one heme-d(1), the former being the electron uptake site while the latter is the nitrite reduction site. Hence, internal electron transfer between these sites is an inherent element in the catalytic cycle of this enzyme. We have investigated the internal electron transfer reaction employing pulse radiolytically produced N-methyl nicotinamide radicals as reductant which reacts solely with the heme-c in an essentially diffusion controlled process. Following this initial step, the reduction equivalent is equilibrating between the c and d(1) heme sites in a unimolecular process (k=23 s(-1), 298 K, pH 7.0) and an equilibrium constant of 1.0. The temperature dependence of this internal electron transfer process has been determined over a 277-313 K temperature range and yielded both equilibrium standard enthalpy and entropy changes as well as activation parameters of the specific rate constants. The significance of these parameters obtained at low degree of reduction of the enzyme is discussed and compared with earlier studies on cd(1) nitrite reductases from other sources.     

Lateral gene transfer of dissimilatory (bi)sulfite reductase revisited

In contrast to previous findings, we demonstrate that the dissimilatory (bi)sulfite reductase genes (dsrAB) of Desulfobacula toluolica were vertically inherited. Furthermore, Desulfobacterium anilini and strain mXyS1 were identified, by dsrAB sequencing of 17 reference strains, as members of the donor lineage for those gram-positive Desulfotomaculum species which laterally acquired dsrAB.     

The location of dissimilatory nitrite reductase and the control of dissimilatory nitrate reductase by oxygen in Paracoccus denitrificans.

1. A method is described for preparing spheroplasts from Paracoccus denitrificans that are substantially depleted of dissimilatory nitrate reductase (cytochrome cd) activity. Treatment of cells with lysozyme + EDTA together with a mild osmotic shock, followed by centrifugation, yielded a pellet of spheroplasts and a supernatant that contained d-type cytochrome. The spheroplasts were judged to have retained an intact plasma membrane on the basis that less than 1% of the activity of a cytoplasmic marker protein, malate dehydrogenase, was released from the spheroplasts. In addition to a low activity towards added nitrite, the suspension of spheroplasts accumulated the nitrite that was produced by respiratory chain-linked reduction of nitrate. It is concluded that nitrate reduction occurs at the periplasmic side of the plasma membrane irrespective of whether nitrite is generated by nitrate reduction or is added exogenously. 2. Further evidence for the integrity of the spheroplasts was that nitrate reduction was inhibited by O2, and that chlorate was reduced at a markedly lower rate than nitrate. These data are taken as evidence for an intact plasma membrane because it was shown that cells acquire the capability to reduce nitrate under aerobic conditions after addition of low amounts of Triton X-100 which, with the same titre, also overcame the permeability barrier to chlorate reduction by intact cells. The close relationship between the appearance of chlorate reduction and the loss of the inhibitory effect of O2 on nitrate reduction also suggests that the later feature of nitrate respiration is due to a control on the accessibility of nitrate to its reductase rather than on the flow of electrons to nitrate reductase.     

Membrane-associated, dissimilatory nitrite reductase of the denitrifying fungus Cylindrocarpon tonkinense

Dissimilatory nitrite reductase (Nir) of the fungus Cylindrocarpon tonkinense was isolated and partially characterized. Nir activity was recovered in both the soluble and the membrane fractions, giving a specific activity of 4.74 and 5.80 μmol NO min^–1 (mg protein)^–1, respectively. The soluble and membrane-associated Nir preparations resembled each other in properties, and the results suggested that Nir is a homodimer of a 67-kDa subunit. The absorption spectrum and the inhibition by diethyldithiocarbamate indicated that fungal Nir is a copper-containing Nir (CuNir). Received: 7 September 1998 / Accepted: 1 December 1998     

Purification of Pseudomonas cytochrome oxidase (or nitrite reductase) by immunological methods

A new purification procedure for the cytochrome oxidase from Pseudomonas aeruginosa based on immunoaffinity chromatography has been compared with the biochemical method and shown to be (i) fully competitive in terms of chemical homogeneity and enzymatic properties of the purified protein (ii) slightly less efficient in terms of total recovery and (iii) much more convenient in terms of the time required. A further evolution of the method that minimizes the number of purification steps and any stress to the native structure of the protein is suggested.     

Respiratory detoxification of nitric oxide by the cytochrome c nitrite reductase of Escherichia coli

Nitric oxide is a key element in host defense against invasive pathogens. The periplasmic cytochrome c nitrite reductase (NrfA) of Escherichia coli catalyzes the respiratory reduction of nitrite, but in vitro studies have shown that it can also reduce nitric oxide. The physiological significance of the latter reaction in vivo has never been assessed. In this study the reduction of nitric oxide by Escherichia coli was measured in strains active or deficient in periplasmic nitrite reduction. Nrf(+) cells, harvested from cultures grown anaerobically, possessed a nitric-oxide reductase activity with physiological electron donation of 60 nmol min(-1) x mg dry wt(-1), and an in vivo turnover number of NrfA of 390 NO* s(-1) was calculated. Nitric-oxide reductase activity could not be detected in Nrf(-) strains. Comparison of the anaerobic growth of Nrf(+) and Nrf(-) strains revealed a higher sensitivity to nitric oxide in the NrfA(-) strains. A higher sensitivity to the nitrosating agent S-nitroso-N-acetyl penicillamine (SNAP) was also observed in agar plate disk-diffusion assays. Oxygen respiration by E. coli was also more sensitive to nitric oxide in the Nrf(-) strains compared with the Nrf(+) parent strain. The results demonstrate that active periplasmic cytochrome c nitrite reductase can confer the capacity for nitric oxide reduction and detoxification on E. coli. Genomic analysis of many pathogenic enteric bacteria reveals the presence of nrf genes. The present study raises the possibility that this reflects an important role for the cytochrome c nitrite reductase in nitric oxide management in oxygen-limited environments.     

Quantum mechanical interpretation of nitrite reduction by cytochrome cd1 nitrite reductase from Paracoccus pantotrophus

The reduction of nitrite to nitric oxide in respiratory denitrification is catalyzed by a cytochrome cd(1) nitrite reductase in Paracoccus pantotrophus (formerly known as Thiosphaera pantotropha LMD 92.63). High-resolution structures are available for the fully oxidized [Fül?p, V., Moir, J. W., Ferguson, S. J., and Hajdu, J. (1995) Cell 81, 369-377; Baker, S. C., Saunders, N. F., Willis, A. C., Ferguson, S. J., Hajdu, J., and Fül?p, V. (1997) J. Mol. Biol. 269, 440-455] and fully reduced forms of this enzyme, as well as for various intermediates in its catalytic cycle [Williams, P. A., Fül?p, V., Garman, E. F., Saunders, N. F., Ferguson, S. J., and Hajdu, J. (1997) Nature 389, 406-412]. On the basis of these structures, quantum mechanical techniques (QM), including density functional methods (DFT), were combined with simulated annealing (SA) and molecular mechanics techniques (MM) to calculate the electronic distribution of molecular orbitals in the active site during catalysis. The results show likely trajectories for electrons, protons, substrates, and products in the process of nitrite reduction, and offer an interpretation of the reaction mechanism. The calculations indicate that the redox state of the d(1) heme and charges on two histidines in the active site orchestrate catalysis locally. Binding of nitrite to the reduced iron is followed by proton transfer from His345 and His388 to one of the oxygens of nitrite, creating a water molecule and an [Fe(II)-NO(+)] complex. Valence isomerization within this complex gives [Fe(III)-NO]. The release of NO from the ferric iron is influenced by the protonation state of His345 and His388, and by the orientation of NO on the d(1) heme. Return of Tyr25 to a hydrogen-bonding position between His345 and His388 facilitates product release, but a rebinding of Tyr25 to the oxidized iron may be bypassed in steady-state catalysis.     

Purification and properties of a dissimilatory nitrite reductase of a denitrifying phototrophic bacterium

Nitrite reductase [nitric-oxide : (acceptor) oxidoreductase,EC 1.7.2.1 [EC] ] from a denitrifying phototrophic bacterium, Rhodopseudomonassphaeroides forma sp. denitrificans, was purified. The molecularweight of the enzyme, estimated by gel-filtration, was 80,000.Sodium dodecyl sulfate polyacrylamide gel electrophoresis ofthe purified enzyme showed a single 39,000 molecular weightband, indicating that the enzyme was composed of two subunitsof identical molecular weight. The oxidized form of the enzymeexhibited maximum absorption at 280 nm, 450 nm and 590 nm, andthe reduced form only at 280 nm. The ESR spectrum of a frozensolution of the oxidized enzyme showed a typical spectrum patternof a copper protein, suggesting that two types of Cu²⁺ existedwithin the enzyme. Estimates with an atomic absorption spectrophotometer,revealed two copper atoms per molecule. The optimum pH of theenzyme was 7.0. Km for nitrite was estimated to be 51 µM,and the optimum temperature, 30?C. The enzyme was inhibitedby CO, potassium cyanide and diethyldithiocarbamate and activatedby monoiodoacetate. Phenazine methosulfate, 2,6-dichlorophenolindophenol,horse heart cytochrome c, and cytochrome c₂ from this bacteriumwere suitable electron donors. The enzyme also showed cytochromec oxidase activity. (Received May 4, 1978; )     

The tetraheme cytochrome c NrfH is required to anchor the cytochrome c nitrite reductase (NrfA) in the membrane of Wolinella succinogenes.

The electron-transport chain that catalyzes nitrite respiration with formate in Wolinella succinogenes consists of formate dehydrogenase, menaquinone and the nitrite reductase complex. The latter catalyzes nitrite reduction by menaquinol and is made up of NrfA and NrfH, two c-type cytochromes. NrfA is the catalytic subunit; its crystal structure is known. NrfH belongs to the NapC/NirT family of membrane-bound c-type cytochromes and mediates electron transport between menaquinol and NrfA. It is demonstrated here by MALDI MS that four heme groups are attached to NrfH. A Delta nrfH deletion mutant of W. succinogenes was constructed by replacing the nrfH gene with a kanamycin-resistance gene cartridge. This mutant did not form the NrfA protein, probably because of a polar effect of the mutation on nrfA expression. The nrfHAIJ gene cluster was restored by integration of an nrfH-containing plasmid into the genome of the Delta nrfH mutant. The resulting strain had wild-type properties with respect to growth by nitrite respiration and nitrite reductase activity. A mutant (stopH) that contained the nrfHAIJ locus with nrfH modified by two artificial stop codons near its 5' end produced wild-type amounts of NrfA in the absence of the NrfH protein. NrfA was located exclusively in the soluble cell fraction of the stopH mutant, indicating that NrfH acts as the membrane anchor of the NrfHA complex in wild-type bacteria. The stopH mutant did not grow by nitrite respiration and did not catalyze nitrite reduction by formate, indicating that the electron transport is strictly dependent on NrfH. The NrfH protein seems to be an unusual member of the NapC/NirT family as it forms a stable complex with its redox partner protein NrfA.     

Pentahaem cytochrome c nitrite reductase: reaction with hydroxylamine,a potential reaction intermediate and substrate

The pentahaem enzyme cytochrome c nitrite reductase catalyses the reduction of nitrite to ammonia, a key reaction in the biological nitrogen cycle. The enzyme can also transform nitrogen monoxide and hydroxylamine, two potential bound reaction intermediates, into ammonia. Structural and mechanistic aspects of the multihaem enzyme are discussed in comparison with hydroxylamine oxidoreductase, a trimeric protein with eight haem molecules per subunit.     

Immunochemical patterns of distribution of nitrous oxide reductase and nitrite reductase (cytochrome cd 1) among denitrifying pseudomonads

The novel multicopper enzyme nitrous oxide reductase from Pseudomonas perfectomarina was purified to homogeneity to study its properties and distribution in various pseudomonads and other selected denitrifying genera by immunochemical techniques. Quantitation of immunochemical crossreactivity by micro-complement fixation within the denitrifying pseudomonads of Palleroni's ribosomal ribonucleic acid group I corresponded to the taxonomic positions established by nucleic acid hybridization. The assignment of P. perfectomarina to the stutzeri-group (as strain ZoBell) was consolidated by immunochemical crossreactivity based on nitrous oxide reductase. Crossreactivity of nitrite reductase (cytochrome cd ₁) with a respective P. perfectomarina rabbit antiserum was limited to strain DSM 50227 of P. stutzeri; although it could not contribute information towards broader relationships within rRNA group I, it lent further prove to the unity of these two species.     

Need for cytochrome bc1 complex for dissimilatory nitrite reduction of Pseudomonas aeruginosa

Pseudomonas aeruginosa strains deficient in the genes for cytochrome c1, a subunit of the cytochrome bc1 complex, or the tetraheme membrane protein NapC, which is similar to NirT of Pseudomonas stutzeri, were constructed and their growth was investigated. The cytochrome c1 mutant could not grow under anaerobic conditions with nitrite as an electron acceptor and did not reduce nitrite in spite of its producing active nitrite reductase. NirM (cytochrome c551) and azurin, which are the direct electron donors for nitrite reductase, were reduced by succinate in the presence of the membrane fraction from the wild-type strain as a mediator but not in the presence of that from the cytochrome c1 mutant. These results indicated that cytochrome bc1 complex was necessary for electron transfer from the membrane quinone pool to nitrite reductase. The NapC mutant grew anaerobically at the expense of nitrite, indicating that NapC was not necessary for nitrite reduction.     

Cardiolipin modulates allosterically the nitrite reductase activity of horse heart cytochrome c

Upon cardiolipin (CL) liposomes binding, horse heart cytochrome c (cytc) changes its tertiary structure disrupting the heme-Fe-Met80 distal bond, reduces drastically the midpoint potential, binds CO and NO with high affinity, displays peroxidase activity, and facilitates peroxynitrite isomerization. Here, the effect of CL liposomes on the nitrite reductase activity of ferrous cytc (cytc-Fe(II)) is reported. In the absence of CL liposomes, hexa-coordinated cytc-Fe(II) displays a very low value of the apparent second-order rate constant for the NO₂ ^?-mediated conversion of cytc-Fe(II) to cytc-Fe(II)-NO (k _on = (7.3 ± 0.7) × 10^?2 M^?1 s^?1; at pH 7.4 and 20.0 °C). However, CL liposomes facilitate the NO₂ ^?-mediated nitrosylation of cytc-Fe(II) in a dose-dependent manner inducing the penta-coordination of the heme-Fe(II) atom. The value of k _on for the NO₂ ^?-mediated conversion of CL-cytc-Fe(II) to CL-cytc-Fe(II)-NO is 2.6 ± 0.3 M^?1 s^?1 (at pH 7.4 and 20.0 °C). Values of the apparent dissociation equilibrium constant for CL liposomes binding to cytc-Fe(II) are (2.2 ± 0.2) × 10^?6 M, (1.8 ± 0.2) × 10^?6 M, and (1.4 ± 0.2) × 10^?6 M at pH 6.5, 7.4, and 8.1, respectively, and 20.0 °C. These results suggest that the NO₂ ^?-mediated conversion of CL-cytc-Fe(II) to CL-cytc-Fe(II)-NO could play anti-apoptotic effects impairing lipid peroxidation and therefore the initiation of the cell death program by the release of pro-apoptotic factors (including cytc) in the cytoplasm.