Membrane tetraheme cytochrome c(m552) of the ammonia-oxidizing nitrosomonas europaea: a ubiquinone reductase

Cytochrome c(m552) (cyt c(m552)) from the ammonia-oxidizing Nitrosomonas europaea is encoded by the cycB gene, which is preceded in a gene cluster by three genes encoding proteins involved in the oxidation of hydroxylamine: hao, hydroxylamine oxidoreductase; orf2, a putative membrane protein; cycA, cyt c(554). By amino acid sequence alignment of the core tetraheme domain, cyt c(m552) belongs to the NapC/TorC family of tetra- or pentaheme cytochrome c species involved in electron transport from membrane quinols to a variety of periplasmic electron shuttles leading to terminal reductases. However, cyt c(m552) is thought to reduce quinone with electrons originating from HAO. In this work, the tetrahemic 27 kDa cyt c(m552) from N. europaea was purified after extraction from membranes using Triton X-100 with subsequent exchange into n-dodecyl beta-d-maltoside. The cytochrome had a propensity to form strong SDS-resistant dimers likely mediated by a conserved GXXXG motif present in the putative transmembrane segment. Optical spectra of the ferric protein contained a broad ligand-metal charge transfer band at approximately 625 nm indicative of a high-spin heme. Mossbauer spectroscopy of the reduced (57)Fe-enriched protein revealed the presence of high-spin and low-spin hemes in a 1:3 ratio. Multimode EPR spectroscopy of the native state showed signals from an electronically interacting high-spin/low-spin pair of hemes. Upon partial reduction, a typical high-spin heme EPR signal was observed. No EPR signals were observed from the other two low-spin hemes, indicating an electronic interaction between these hemes as well. UV-vis absorption data indicate that CO (ferrous enzyme) or CN(-) (ferric or ferrous enzyme) bound to more than one and possibly all hemes. Other anionic ligands did not bind. The four ferrous hemes of the cytochrome were rapidly oxidized in the presence of oxygen. Comparative modeling, based on the crystal structure and conserved residues of the homologous NrfH protein from Desulfovibrio of cyt c(m552), predicted some structural elements, including a Met-ligated high-spin heme in a quinone-binding pocket, and likely axial ligands to all four hemes.     

Redox equilibria in hydroxylamine oxidoreductase. Electrostatic control of electron redistribution in multielectron oxidative processes

We report results of continuum electrostatics calculations of the cofactor redox potentials, and of the titratable group pK(a) values, in hydroxylamine oxidoreductase (HAO). A picture of a sophisticated multicomponent control of electron flow in the protein emerged from the studies. First, we found that neighboring heme cofactors strongly interact electrostatically, with energies of 50-100 mV. Thus, cofactor redox potentials depend on the oxidation state of other cofactors, and cofactor redox potentials in the active (partially oxidized) enzyme differ substantially from the values obtained in electrochemical redox titration experiments. We found that, together, solvent-exposed heme 1 (having a large negative redox potential) and heme 2 (having a large positive redox potential) form a lock for electrons generated during the oxidation reaction The attachment of HAO's physiological electron transfer partner cytochrome c(554) results in a positive shift in the redox potential of heme 1, and "opens the electron gate". Electrons generated as a result of hydroxylamine oxidation travel to heme 3 and heme 8, which have redox potentials close to 0 mV versus NHE (this result is in partial disagreement with an existing experimental redox potential assignment). The closeness of hemes 3 and 8 from different enzyme subunits allows redistribution of the four electrons generated as a result of hydroxylamine oxidation, among the three enzyme subunits. For the multielectron oxidation process to be maximally efficient, the redox potentials of the electron-accepting cofactors should be roughly equal, and electrostatic interactions between extra electrons on these cofactors should be minimal. The redox potential assignments presented in the paper satisfy this general rule.     

Cytochrome c peroxidase activity of a protease-modified form of cytochrome c-552 from the denitrifying bacterium Pseudomonas perfectomarina

Protease activity present in aerobically grown cells of Pseudomonas perfectomarina, protease apparently copurified with cytochrome c-552, and trypsin achieved a limited proteolysis of the diheme cytochrome c-552. That partial lysis conferred cytochrome c peroxidase activity upon cytochrome c-552. The removal of a 4000-Da peptide explains the structural changes in the cytochrome c-552 molecule that resulted in the appearance of both cytochrome c peroxidase activity (with optimum activity at pH 8.6) and a high-spin heme iron. The oxidized form of the modified cytochrome c-552 bound cyanide to the high-spin ferric heme with a rate constant of (2.1 +/- 0.1) X 10(3) M-1 s-1. The dissociation constant was 11.2 microM. Whereas the intact cytochrome c-552 molecule can be half-reduced by ascorbate, the cytochrome c peroxidase was not reducible by ascorbate, NADH, ferrocyanide, or reduced azurin. Dithionite reduced the intact protein completely but only half-reduced the modified form. The apparent second-order rate constant for dithionite reduction was (7.1 +/- 0.1) X 10(2) M-1 s-1 for the intact protein and (2.2 +/- 0.1) X 10(3) M-1 s-1 for the modified form. In contrast with other diheme cytochrome c peroxidases, reduction of the low-spin heme was not necessary to permit ligand binding by the high-spin heme iron.     

Spectroscopic characterization of the NO adduct of hydroxylamine oxidoreductase

Hydroxylamine oxidoreductase (HAO) from the autotrophic nitrifying bacterium Nitrosomonas europaea catalyzes the oxidation of NH2OH to NO2-. The enzyme contains eight hemes per subunit which participate in catalysis and electron transport. NO is found to bind to the enzyme and inhibit electron flow to the acceptor protein, cytochrome c554. NO is found to oxidize either partially or fully reduced HAO, but NO will not reduce ferric HAO. Since NO can be reduced but not oxidized to product by HAO, NO is not considered to be a long-lived intermediate in the catalytic mechanism. Substrate oxidation occurs in the presence of bound NO or cyanide, suggesting a second interaction site for substrate with HAO and providing a means for recovery of the NO-inhibited form of the enzyme. Upon addition of NO to oxidized HAO, the integer-spin EPR signal from the active site vanishes, an IR band from NO appears at 1920 cm(-1), and a diamagnetic quadrupole iron doublet appears in M?ssbauer spectroscopy with delta = 0.06 mm/s and DeltaEq = 2.1 mm/s. The NO stretching frequency and M?ssbauer parameters are characteristic of an [FeNO]6 heme complex. New M?ssbauer data on ferric myoglobin-NO are also presented for comparison. The results indicate that NO binds to heme P460 and that the loss of the integer-spin EPR signal is due to the conversion of heme P460 to a diamagnetic S = 0 state and concomitant loss of magnetic interaction with neighboring heme 6. In previous studies where the heme P460-heme 6 interaction was affected by substrate or cyanide binding, a signal attributable to heme 6 was not observable. In contrast, in this work, the NO-induced loss of the signal is accompanied by the appearance of a previously unobserved large g(max) (or HALS) low-spin EPR signal from heme 6.     

High-resolution structures of the oxidized and reduced states of cytochrome c554 from Nitrosomonas europaea

Cytochrome c554 (cyt c554) is a tetra-heme cytochrome involved in the oxidation of NH3 by Nitrosomonas europaea. The X-ray crystal structures of both the oxidized and dithionite-reduced states of cyt c554 in a new, rhombohedral crystal form have been solved by molecular replacement, at 1.6 A and 1.8 A resolution, respectively. Upon reduction, the conformation of the polypeptide chain changes between residues 175 and 179, which are adjacent to hemes III and IV. Cyt c554 displays conserved heme-packing motifs that are present in other heme-containing proteins. Comparisons to hydroxylamine oxidoreductase, the electron donor to cyt c554, and cytochrome c nitrite reductase, an enzyme involved in nitrite ammonification, reveal substantial structural similarity in the polypeptide chain surrounding the heme core environment. The structural determinants of these heme-packing motifs extend to the buried water molecules that hydrogen bond to the histidine ligands to the heme iron. In the original structure determination of a tetragonal crystal form, a cis peptide bond between His129 and Phe130 was identified that appeared to be stabilized by crystal contacts. In the rhombohedral crystal form used in the present high-resolution structure determination, this peptide bond adopts the trans conformation, but with disallowed angles of phi and psi.     

Molecular biology and biochemistry of ammonia oxidation by Nitrosomonas europaea

Nitrosomonas europaea uses only NH(3), CO(2) and mineral salts for growth and as such it is an obligate chemo-lithoautotroph. The oxidation of NH(3) is a two-step process catalyzed by ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO). AMO catalyzes the oxidation of NH(3) to NH(2)OH and HAO catalyzes the oxidation of NH(2)OH to NO(2)(-). AMO is a membrane-bound enzyme composed of three subunits. HAO is located in the periplasm and is a homotrimer with each subunit containing eight c-type hemes. The electron flow from HAO is channeled through cytochrome c(554) to cytochrome c(m552), where it is partitioned for further utilization. Among the ammonia-oxidizing bacteria, the genes for AMO, these cytochromes, and HAO are present in up to three highly similar copies. Mutants with mutations in the copies of amoCAB and hao in N. europaea have been isolated. All of the amoCAB and hao gene copies are functional. N. europaea was selected by the United States Department of Energy for a whole-genome sequencing project. In this article, we review recent research on the molecular biology and biochemistry of NH(3) oxidation in nitrifiers.     

Heme P460 of hydroxylamine oxidoreductase of Nitrosomonas. Reaction with CO and H2O2

Hydroxylamine oxidoreductase (HAO) of Nitrosomonas catalyzes the dehydrogenation of NH2OH and subsequent addition of oxygen to form nitrite. HAO contains c hemes and the CO-binding heme P460 in a 7:1 ratio; dehydrogenation of NH2OH involves passage of electrons to P460 and then c hemes. We now report that electrons rapidly pass from c hemes of HAO to the P460 center and then to H2O2. This conclusion is supported by (a) inhibition of c heme oxidation with CO and (b) loss of H2O2-oxidizability of ferrous c hemes following specific destruction of heme P460. Reaction of ferrous P460 with H2O2 is rate-limiting. Activation of dioxygen for N-oxidation by ferrous HAO may involve the two-electron reduction of O2 by P460. The reaction of ferrous HAO with H2O2 was studied as it may reveal aspects of the mechanism of activation of dioxygen. Reaction of ferrous heme P460 with CO is slow and with low affinity as compared with other hemoproteins. Values for reaction of CO with enzyme were: k1, 1.1 X 10(-3) M-1 s-1 and Kd, 12 microM.     

Protective effect of L-carnitine on hyperammonemia

The diheme cytochrome c-554 which participates in ammonia oxidation in the chemoautotroph , Nitrosomonas europaea has been studied by Soret excitation resonance Raman spectroscopy. The Raman spectrum of reduced cytochrome c-554 at neutral pH is similar classical 6-coordinate low-spin ferrous mammalian cytochrome c. In contrast, the spectrum of ferric cytochrome c-554 suggests a 5-coordinate state which is unusual for c hemes. The oxidized spectrum closely resemble that of horseradish peroxidase (HRP) or cytochrome c peroxidase (CcP) at pH 6.4. The narrow linewidth of the heme core-size vibrations indicates that both heme irons of c-554 have similar geometries.     

Structure and sequence conservation of hao cluster genes of autotrophic ammonia-oxidizing bacteria: evidence for their evolutionary history

Comparison of the organization and sequence of the hao (hydroxylamine oxidoreductase) gene clusters from the gammaproteobacterial autotrophic ammonia-oxidizing bacterium (aAOB) Nitrosococcus oceani and the betaproteobacterial aAOB Nitrosospira multiformis and Nitrosomonas europaea revealed a highly conserved gene cluster encoding the following proteins: hao, hydroxylamine oxidoreductase; orf2, a putative protein; cycA, cytochrome c(554); and cycB, cytochrome c(m)(552). The deduced protein sequences of HAO, c(554), and c(m)(552) were highly similar in all aAOB despite their differences in species evolution and codon usage. Phylogenetic inference revealed a broad family of multi-c-heme proteins, including HAO, the pentaheme nitrite reductase, and tetrathionate reductase. The c-hemes of this group also have a nearly identical geometry of heme orientation, which has remained conserved during divergent evolution of function. High sequence similarity is also seen within a protein family, including cytochromes c(m)(552), NrfH/B, and NapC/NirT. It is proposed that the hydroxylamine oxidation pathway evolved from a nitrite reduction pathway involved in anaerobic respiration (denitrification) during the radiation of the Proteobacteria. Conservation of the hydroxylamine oxidation module was maintained by functional pressure, and the module expanded into two separate narrow taxa after a lateral gene transfer event between gamma- and betaproteobacterial ancestors of extant aAOB. HAO-encoding genes were also found in six non-aAOB, either singly or tandemly arranged with an orf2 gene, whereas a c(554) gene was lacking. The conservation of the hao gene cluster in general and the uniqueness of the c(554) gene in particular make it a suitable target for the design of primers and probes useful for molecular ecology approaches to detect aAOB.     

Reduction of cytochrome c peroxidase compounds I and II by ferrocytochrome c. A stopped-flow kinetic investigation

The oxidation of yeast cytochrome c peroxidase by hydrogen peroxide produces a unique enzyme intermediate, cytochrome c peroxidase Compound I, in which the ferric heme iron has been oxidized to an oxyferryl state, Fe(IV), and an amino acid residue has been oxidized to a radical state. The reduction of cytochrome c peroxidase Compound I by horse heart ferrocytochrome c is biphasic in the presence of excess ferrocytochrome c as cytochrome c peroxidase Compound I is reduced to the native enzyme via a second enzyme intermediate, cytochrome c peroxidase Compound II. In the first phase of the reaction, the oxyferryl heme iron in Compound I is reduced to the ferric state producing Compound II which retains the amino acid free radical. The pseudo-first order rate constant for reduction of Compound I to Compound II increases with increasing cytochrome c concentration in a hyperbolic fashion. The limiting value at infinite cytochrome c concentration, which is attributed to the intracomplex electron transfer rate from ferrocytochrome c to the heme site in Compound I, is 450 +/- 20 s-1 at pH 7.5 and 25 degrees C. Ferricytochrome c inhibits the reaction in a competitive manner. The reduction of the free radical in Compound II is complex. At low cytochrome c peroxidase concentrations, the reduction rate is 5 +/- 3 s-1, independent of the ferrocytochrome c concentration. At higher peroxidase concentrations, a term proportional to the square of the Compound II concentration is involved in the reduction of the free radical. Reduction of Compound II is not inhibited by ferricytochrome c. The rates and equilibrium constant for the interconversion of the free radical and oxyferryl forms of Compound II have also been determined.     

Two structurally and kinetically distinct forms of Wolinella succinogenes nitrite reductase.

It is shown that the oxidized form of the hexa-haem nitrite reductase of Wolinella succinogenes exists in two structurally and functionally distinct forms, termed 'resting' and 'redox-cycled'. The nitrite reductase as initially isolated, termed 'resting', has five low-spin ferrihaem groups and one high-spin ferrihaem group. The reduction of these haem groups by Na2S2O4 occurs in two kinetically and spectrally distinct phases. In the slower phase the haem groups are reduced by dithionite with a limiting rate of 4 s-1. If the enzyme is re-oxidized after reduction with dithionite or with methyl viologen, the resulting ferric form, termed 'redox-cycled', possesses only low-spin haem centres and a rate of reduction in the slower phase that is no longer limited. In the resting form of the enzyme the high-spin ferrihaem group is weakly exchange-coupled to a low-spin haem group. It is proposed that in the redox-cycled form the exchange coupling occurs between two low-spin ferric haem groups. This change in spin state allows a more rapid rate of electron transfer to the coupled pair.     

Ionic strength dependence of the kinetics of electron transfer from bovine mitochondrial cytochrome c to bovine cytochrome c oxidase.

The effect of ionic strength on the one-electron reduction of oxidized bovine cytochrome c oxidase by reduced bovine cytochrome c has been studied by using flavin semiquinone reductants generated in situ by laser flash photolysis. In the absence of cytochrome c, direct reduction of the heme a prosthetic group of the oxidase by the one-electron reductant 5-deazariboflavin semiquinone occurred slowly, despite a driving force of approximately +1 V. This is consistent with a sterically inaccessible heme a center. This reduction process was independent of ionic strength from 10 to 100 mM. Addition of cytochrome c resulted in a marked increase in the amount of reduced oxidase generated per laser flash. Reduction of the oxidase at the heme a site was monophasic, whereas oxidation of cytochrome c was multiphasic, the fastest phase corresponding in rate constant to the reduction of the heme a. During the fast kinetic phase, 2 equiv of cytochrome c was oxidized per heme a reduced. We presume that the second equivalent was used to reduce the Cua center, although this was not directly measured. The first-order rate-limiting process which controls electron transfer to the heme a showed a marked ionic strength effect, with a maximum rate constant occurring at mu = 110 mM (1470 s-1), whereas the rate constant obtained at mu = 10 mM was 630 s-1 and at mu = 510 mM was 45 s-1. There was no effect of "pulsing" the enzyme on this rate-limiting one-electron transfer process. These results suggest that there are structural differences in the complex(es) formed between mitochondrial cytochrome c and cytochrome c oxidase at very low and more physiologically relevant ionic strengths, which lead to differences in electron-transfer rate constants.     

Cytochrome oxidase from Pseudomonas aeruginosa. IV. Reaction with oxygen and carbon monoxide.

The reaction between a cytochrome oxidase from Pseudomonas aeruginosa and oxygen has been studied by a rapid mixing technique. The data indicate that the heme d1 moiety of the ascorbate-reduced enzyme is oxidized faster than the heme c component. The oxidation of heme d1 is accurately second order with respect to oxygen and has a rate constant of 5.7 - 10(4) M-1 - s-1 at 20 degrees C. The oxidation of the heme c has a first order rate constant of about 8 s-1 at infinite concentration of O2. The results indicate that the rate-limiting step is the internal transfer of electrons from heme c to heme d1. These more rapid reactions are followed by more complicated but smaller abcorbance changes whose origin is still not clear. The reaction of ascorbate-reduced oxidase with CO has also been studied and is second order with a rate constant of 1.8 - 10(4) M-1 - s-1. The initial reaction with CO is followed by a slower reaction of significantly less magnitude. The equilibrium constant for the reaction with CO, calculated as a dissociation constant from titrimetric experiments with dithionite-reduced oxidase, is about 2.3 - 10(-6) M. From these data a rate constant of 0.041 s-1 can be calculated for the dissociation of CO from the enzyme.     

Spin-equilibrium and heme-ligand alteration in a high-potential monoheme cytochrome (cytochrome c554) from Achromobacter cycloclastes, a denitrifying organism

A c-type monoheme cytochrome c554 (13 kDa) was isolated from cells of Achromobacter cycloclastes IAM 1013 grown anaerobically as a denitrifier. The visible absorption spectrum indicates the presence of a band at 695 nm characteristic of heme-methionine coordination (low-spin form) coexisting with a minor high-spin form as revealed by the contribution at 630 nm. Magnetic susceptibility measurements support the existence of a small contribution of a high-spin form at all pH values, attaining a minimum at intermediate pH values. The mid-point redox potential determined by visible spectroscopy at pH 7.2 is +150 mV. The pH-dependent spin equilibrum and other relevant structural features were studied by 300-MHz 1H-NMR spectroscopy. In the oxidized form, the 1H-NMR spectrum shows pH dependence with pKa values at 5.0 and 8.9. According to these pKa values, three forms designated as I, II and III can be attributed to cytochrome c554. Forms I and II predominate at low pH values, and the 1H-NMR spectra reveal heme methyl proton resonances between 40 ppm and 22 ppm. These forms have a methionyl residue as a sixth ligand, and C6 methyl group of the bound methionine was identified in the low-field region of the NMR spectra. Above pH 9.6, form III predominates and the 1H-NMR spectrum is characterized by down-field hyperfine-shifted heme methyl proton resonances between 29 ppm and 22 ppm. Two new resonances are observed at congruent to 66 ppm and 54 ppm, and are taken as indicative of a new type of heme coordination (probably a lysine residue). These pH-dependent features of the 1H-NMR spectra are discussed in terms of the heme environment structure. The chemical shifts of the methyl resonances at different pH values exhibit anti-Curie temperature dependence. In the ferrous state, the 1H-NMR spectrum shows a methyl proton resonance at -3.9 ppm characteristic of methionine axial ligation. The electron-transfer rate between ferric and ferrous forms has been estimated to be smaller than 2 x 10(4) M-1 s-1 at pH 5. EPR spectroscopy was also used to probe the ferric heme environment. A prominent signal at gmax congruent to 3.58 and the overall lineshape of the spectrum indicate an almost axial heme environment.     

Multiple copies of genes coding for electron transport proteins in the bacterium Nitrosomonas europaea.

The genome of Nitrosomonas europaea contains at least three copies each of the genes coding for hydroxylamine oxidoreductase (HAO) and cytochrome c554. A copy of an HAO gene is always located within 2.7 kb of a copy of a cytochrome c554 gene. Cytochrome P-460, a protein that shares very unusual spectral features with HAO, was found to be encoded by a gene separate from the HAO genes.     

Equilibrium unfolding of a small low-potential cytochrome, cytochrome c553 from Desulfovibrio vulgaris.

To understand general aspects of stability and folding of c-type cytochromes, we have studied the folding characteristics of cytochrome c553 from Desulfovibrio vulgaris (Hildenborough). This cytochrome is structurally similar but lacks sequence homology to other heme proteins; moreover, it has an abnormally low reduction potential. Unfolding of oxidized and reduced cytochrome c553 by guanidine hydrochloride (GuHCl) was monitored by circular dichroism (CD) and Soret absorption; the same unfolding curves were obtained with both methods supporting that cytochrome c553 unfolds by an apparent two-state process. Reduced cytochrome c553 is 7(3) kJ/mol more stable than the oxidized form; accordingly, the reduction potential of unfolded cytochrome c553 is 100(20) mV more negative than that of the folded protein. In contrast to many other unfolded cytochrome c proteins, upon unfolding at pH 7.0 both oxidized and reduced heme in cytochrome c553 become high-spin. The lack of heme misligation in unfolded cytochrome c553 implies that its unfolded structure is less constrained than those of cytochromes c with low-spin, misligated hemes.     

Active site structure of SoxB-type cytochrome bo3 oxidase from thermophilic Bacillus

Two-subunit SoxB-type cytochrome c oxidase in Bacillus stearothermophilus was over-produced, purified, and examined for its active site structures by electron paramagnetic resonance (EPR) and resonance Raman (RR) spectroscopies. This is cytochrome bo3 oxidase containing heme B at the low-spin heme site and heme O at the high-spin heme site of the binuclear center. EPR spectra of the enzyme in the oxidized form indicated that structures of the high-spin heme O and the low-spin heme B were similar to those of SoxM-type oxidases based on the signals at g=6.1, and g=3.04. However, the EPR signals from the CuA center and the integer spin system at the binuclear center showed slight differences. RR spectra of the oxidized form showed that heme O was in a 6-coordinated high-spin (nu3 = 1472 cm(-1)), and heme B was in a 6-coordinated low-spin (nu3 = 1500 cm(-1)) state. The Fe2+-His stretching mode was observed at 211 cm(-1), indicating that the Fe2+-His bond strength is not so much different from those of SoxM-type oxidases. On the contrary, both the Fe2+-CO stretching and Fe2+-C-O bending modes differed distinctly from those of SoxM-type enzymes, suggesting some differences in the coordination geometry and the protein structure in the proximity of bound CO in cytochrome bo3 from those of SoxM-type enzymes.     

Coordination and redox properties of a novel triheme cytochrome from Desulfovibrio vulgaris (Hildenborough)

A high molecular weight multiheme c-type cytochrome from the sulfate-reducing bacterium Desulfovibrio vulgaris (Hildenborough) has been spectroscopically characterized and compared with the tetraheme cytochrome c3. The protein contains a pentacoordinate high-spin heme (gz 6.0) and two hexacoordinate low-spin hemes (gz 2.95, gy 2.27, gx 1.48). From analysis of the g values for the low-spin hemes by the procedure of Blumberg and Peisach (Palmer, 1983) and comparison with with the optical spectra from a variety of c-type cytochromes, it is likely that these low-spin hemes are bound by two histidine residues. The NO derivative displayed typical rhombic EPR features (gx 2.07, gz 2.02, gy 1.99). Addition of azide does not lead to coupling between heme chromophores, but the ligand is accessible to the high-spin heme. The use of a glassy-carbon electrode to perform direct (no promoter) electrochemistry on the cytochrome is illustrated. Differential pulse polarography of the native protein gave two waves with reduction potentials of -59 (5) and -400 (8) mV (versus NHE). The cyanide adduct gave two waves with reduction potentials of -263 (8) and -401 (8) mV. The cytochrome was found to catalyze the reduction of nitrite and hydroxylamine.     

Electron transfer during the oxidation of ammonia by the chemolithotrophic bacterium Nitrosomonas europaea

The combined action of ammonia monooxygenase, AMO, (NH(3)+2e(-)+O(2)-->NH(2)OH) and hydroxylamine oxidoreductase, HAO, (NH(2)OH+H(2)O-->HNO(2)+4e(-)+4H(+)) accounts for ammonia oxidation in Nitrosomonas europaea. Pathways for electrons from HAO to O(2), nitrite, NO, H(2)O(2) or AMO are reviewed and some recent advances described. The membrane cytochrome c(M)552 is proposed to participate in the path between HAO and ubiquinone. A bc(1) complex is shown to mediate between ubiquinol and the terminal oxidase and is shown to be downstream of HAO. A novel, red, low-potential, periplasmic copper protein, nitrosocyanin, is introduced. Possible mechanisms for the inhibition of ammonia oxidation in cells by protonophores are summarized. Genes for nitrite- and NO-reductase but not N(2)O or nitrate reductase are present in the genome of Nitrosomonas. Nitrite reductase is not repressed by growth on O(2); the flux of nitrite reduction is controlled at the substrate level.     

Magnetic susceptibility measurements on Pseudomonas cytochrome cd1

The magnetic susceptibilities of cytochrome cd1 from Pseudomonas aeruginosa (American Type Culture Collection 19429) have been measured by a nuclear magnetic resonance technique. In the oxidized form both heme c and heme d1 are in the low-spin state with an average magnetic moment of 2.6 Bohr magnetons. At 25 degrees C and pH 8.0, the ascorbate-reduced cytochrome contains one low-spin and one high-spin heme per subunit. Based on previous reports in the literature, the high-spin ferrous heme has been assigned to the heme d1 group. At pH 8.0 the ascorbate-reduced heme d1 has a magnetic moment of 5.3 Bohr magnetons. This value decreases to 4.9 at pH 5.5, but is still indicative of a high-spin ferrous system. The paramagnetic susceptibility of the ferricytochrome demonstrated a temperature dependence consistent with Curie's law, but the ferrocytochrome showed an increase in paramagnetic susceptibility with increasing temperature.