Spectroscopic and rapid kinetic studies of reduction of cytochrome c554 by hydroxylamine oxidoreductase from Nitrosomonas europaea.

During oxidation of hydroxylamine, hydroxylamine oxidoreductase (HAO) transfers two electrons to tetraheme cytochrome c554 at rates sufficient to account for physiological rates of oxidation of ammonia to nitrite in Nitrosomonas europaea. Spectroscopic changes indicate that the two electrons are taken up by a high-potential pair of hemes (E degrees' = +47 mV) (one apparently high spin and one low spin). During single-turnover experiments, in which the reduction of oxidized cytochrome c554 by NH2OH-reduced HAO is monitored, one electron is taken up by the high-spin heme at a rate too fast to monitor directly (greater than 100 s-1) but which is inferred either by a loss of amplitude (relative to that observed under multiple-turnover conditions) or is slowed down by increasing ionic strength (greater than or equal to 300 mM KCl). The second electron is taken up by the low-spin heme at a 10-30-fold slower rate. The latter kinetics appear multiphasic and may be complicated by a transient oxidation of HAO due to the rapid transfer of the first electron into the high-spin heme of cytochrome c554. Under multiple-turnover conditions, a "slower" rate of reduction is observed for the high-spin heme of cytochrome c554 with a maximum rate constant of approximately 30 s-1, a value also obtained for the reduction, by NH2OH, of the cytochrome c554 high-spin heme within an oxidized HAO/c554 complex. Under these conditions, the maximum rate of reduction of the low-spin heme was approximately 11.0 s-1. Both rates decreased as the concentration of cytochrome c554 was increased above the concentration of HAO.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Organization of the hao gene cluster of Nitrosomonas europaea: genes for two tetraheme c cytochromes.

The organization of genes for three proteins involved in ammonia oxidation in Nitrosomonas europaea has been investigated. The amino acid sequence of the N-terminal region and four heme-containing peptides produced by proteolysis of the tetraheme cytochrome c554 of N. europaea were determined by Edman degradation. The gene (cycA) encoding this cytochrome is present in three copies per genome (H. McTavish, F. LaQuier, D. Arciero, M. Logan, G. Mundfrom, J.A. Fuchs, and A. B. Hooper, J. Bacteriol. 175:2445-2447, 1993). Three clones, representing at least two copies of cycA, were isolated and sequenced by the dideoxy-chain termination procedure. In both copies, the sequences of 211 amino acids derived from the gene sequence are identical and include all amino acids predicted by the proteolytic peptides. In two copies, the cycA open reading frame (ORF) is followed closely (three bases in one copy) by a second ORF predicted to encode a 28-kDa tetraheme c cytochrome not previously characterized but similar to the nirT gene product of Pseudomonas stutzeri. In one copy of the cycA gene cluster, the second ORF is absent.     

Characterization of a mouse somatic cytochrome c gene and three cytochrome c pseudogenes.

Mouse contains two functional, but differentially expressed, cytochrome c genes. One of these genes is expressed in all somatic tissues so far examined. The other gene is expressed only in testis and is assumed to be spermatogenesis-specific. The nucleotide sequence of four mouse cytochrome c-like genes has been determined. One of these genes (MC1) contains an intron and encodes a polypeptide sequence identical to the published mouse somatic cytochrome c amino acid sequence. The other three genes can not properly encode a mouse cytochrome c protein and appear to be pseudogenes which have arisen via an insertion into the mouse genome of a cDNA copy of a cytochrome c mRNA molecule.     

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 c₅₅₄; and cycB, cytochrome c_m552. The deduced protein sequences of HAO, c₅₅₄, and c_m552 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_m552, 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 c₅₅₄ gene was lacking. The conservation of the hao gene cluster in general and the uniqueness of the c₅₅₄ gene in particular make it a suitable target for the design of primers and probes useful for molecular ecology approaches to detect aAOB.     

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.     

Effect of aerobic growth conditions on the soluble cytochrome content of the purple phototrophic bacterium Rhodobacter sphaeroides: induction of cytochrome c554

When grown anaerobically in the light, Rhodobacter sphaeroides contains appreciable quantities of cytochromes c2 and c', but smaller amounts of other soluble cytochromes such as cytochrome c551.5, cytochrome c554, and an oxygen-binding heme protein. When R. sphaeroides is mass cultured aerobically in the dark to stationary phase, the content of cytochrome c2 does not change appreciably, whereas cytochrome c554 is approximately 8-fold more abundant, cytochrome c' is at least 10-fold less abundant, and cytochrome c551.5 is fivefold lower than in the phototrophically grown cells. These observations confirm previous literature reports that in this organism a cytochrome c553 (or c554 in our experience) is more abundant when cells are grown aerobically. Furthermore, the aerobic cytochrome c554 is positively identified with the previously characterized minor cytochrome c554 component of anaerobic photosynthetic cells. Preliminary sequence results show that cytochrome c554 is a member of the cytochrome c' structural family, but differs from normal cytochromes c' in having a methionine sixth ligand to the heme. The levels of electron carrier proteins of low redox potential had previously been reported to be less in aerobic than in photoheterotrophic cells and we have verified that observation for the specific examples of cytochromes c' and c551.5. The oxygen binding heme protein, SHP, is not induced by aerobic growth.     

The roles of the three gene copies encoding hydroxylamine oxidoreductase in Nitrosomonas europaea

The nitrifying bacterium Nitrosomonas europaea contains three copies of the gene (hao) encoding hydroxylamine oxidoreductase (HAO), the second enzyme in the nitrification pathway which oxidizes NH(2)OH to NO(2)(-). The nucleotide sequences of the hao genes differ by only one nucleotide. Two of the three gene copies have identical promoter sequences, while the third promoter has a different nucleotide sequence. Mutant strains with two of the three copies of hao inactivated were created by insertional inactivation, using DNA cassettes containing kanamycin- and gentamycin-resistance genes. All three double-mutant combinations were obtained. These double mutants were phenotypically identical under the conditions tested. Two of these double mutants were similar to wild-type cells or cells having a single hao copy inactivated regarding growth rates or hydroxylamine-dependent O(2) uptake activity, but had only about 50% of the wild-type level of in vitro HAO activity and hao mRNA. The third hao double mutant had an unstable genotype, resulting in recombination of the gentamycin marker into another copy of hao. The N. europaea genomic sequence was recently completed, revealing the locations of the copies of hao and other nitrification genes. Comparison with the arrangement of hao genes in the closely related strain, Nitrosomonas sp. strain ENI-11, showed a similar organization.     

A partial resolution and reconstitution of the ammonia-oxidizing system of Nitrosomonas europaea: role of cytochrome c554

The cell-free ammonia-oxidizing system of Nitrosomonas europaea was resolved into three major fractions: a membrane fraction containing cytochrome a1 and c-type cytochromes, a fraction with hydroxylamine-cytochrome c reductase and a cytochrome c fraction. The ammonia-oxidizing activity was reconstituted by the combination of these three fractions. The activity was more consistently reconstituted by adding Nitrosomonas cytochrome c554 to the membrane fraction. The hydroxylamine-cytochrome c reductase activity of the membrane fraction increased with the addition of cytochrome c554, but the oxidation of hydroxylamine to nitrite required a further addition of cytochrome c552. The ammonia oxidation by the membrane plus cytochrome c554 was affected by the concentration of phosphate and the addition of bovine serum albumin, spermine, or MgCl2.     

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.     

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 molecular evolutionary analysis of a plant cytochrome c gene: Surprising implications forArabidopsis thaliana

Summary We have isolated a cytochrome c gene fromArabidopsis thaliana (cv. Columbia), which is the first cytochrome c gene to be cloned from a higher plant. Genomic DNA blot analysis indicates that there is only one copy of cytochrome c inArabidopsis. The gene consists of three exons separated by two introns. Gene features such as regulatory regions, codon usage, and conserved splicing-specific sequences are all present and typical of dicotyledonous plant nuclear genes. We have constructed phenograms and cladograms for cytochrome c amino acid sequences and histone H3, alcohol dehydrogenase, and actin DNA sequences. For both cytochrome c and histone H3,Arabidopsis clusters poorly with other higher plants. Instead, it clusters withNeurospora and/or the yeasts. We suggest that perhaps this observation should be considered when usingArabidopsis as a model system for higher plants.     

Evolution of an octahaem cytochrome c protein family that is key to aerobic and anaerobic ammonia oxidation by bacteria

The biogeochemical nitrogen cycle is mediated by many groups of microorganisms that harbour octahaem cytochromes c (OCC). In this study molecular evolutionary analyses and the conservation of predicted functional residues and secondary structure were employed to investigate the descent of OCC proteins related to hydroxylamine oxidoreductase (HAO) and hydrazine oxidoreductase (HZO) from pentahaem cytochrome c nitrite reductase (NrfA). An octahaem cytochrome cnitrite reductase (ONR) was shown to be a possible intermediate in the process. Analysis of genomic neighbourhoods of OCC protein-encoding genes revealed adjacent conserved genes whose products, together with HAO, provide a path of electron transfer to quinone and constitute a functional catabolic module. The latter has evolved more than once under a variety of functional pressures on the catabolic lifestyles of their bacterial hosts. Structurally, the archetypical long helices in the large C-terminal domain of the proteins as well as the distal axial ligands to most haems were highly conserved in NrfA and all descendents. Residues known to be involved in the nitrite reductase activity of NrfA including the 'CxxCK' motif at the catalytic haem, the substrate and Ca binding sites, and the nitrite and ammonium channels were conserved in the eight representatives of ONR. In the latter, a unique cysteine has been inserted above the active site. The 64 other OCC proteins differed from ONR by the absence of the 'CxxCK' motif, the channel residues and most of the Ca-binding residues and the conserved presence of an 'Asp-His' pair inserted above the active site as well as the tyrosine that forms an intersubunit cross-link to the catalytic haem of HAO. Our proposed scenario of evolution of OCC proteins in the HAO family from NrfA is supported by (i) homology based on sequence and structure, (ii) its wide distribution among bacterial taxa, (iii) the dedicated interaction with specific proteins, and it is (iv) congruent with geological history.     

Identification of cDNA encoding cytochrome c oxidase subunit 5c (COX5c) from rice: comparison of its expression with nuclear-encoded and mitochondrial-encoded COX genes

Little is presently known about the nuclear-encoded genes for cytochrome c oxidase (COX) in higher plants. In rice, only the nuclear-encoded COX5b gene has been reported. To understand the relationship between the expression of nuclear-encoded and mitochondrial-encoded COX genes in rice, we first characterized a cDNA encoding one of the other nuclear COX genes, COX5c, which encodes 63 amino acids. The deduced amino acid sequence of COX5c from rice was highly homologous to that from sweet potato. Genomic Southern hybridization indicated that the rice COX5c subunit is encoded by a single copy of the COX5c gene. Furthermore, we compared the expression patterns of the nuclear-encoded COX5c and COX5b genes with the expression pattern of the mitochondrial-encoded COX1 gene among several organs by Northern blot analysis. The results suggested that regulatory systems of expression between the nuclear-encoded and the mitochondrial-encoded COX genes are different among different organs in rice.     

Parallel electron donation pathways to cytochrome c(z) in the type I homodimeric photosynthetic reaction center complex of Chlorobium tepidum

We studied the regulation mechanism of electron donations from menaquinol:cytochrome c oxidoreductase and cytochrome c-554 to the type I homodimeric photosynthetic reaction center complex of the green sulfur bacterium Chlorobium tepidum. We measured flash-induced absorption changes of multiple cytochromes in the membranes prepared from a mutant devoid of cytochrome c-554 or in the reconstituted membranes by exogenously adding cytochrome c-555 purified from Chlorobium limicola. The results indicated that the photo-oxidized cytochrome c(z) bound to the reaction center was rereduced rapidly by cytochrome c-555 as well as by the menaquinol:cytochrome c oxidoreductase and that cytochrome c-555 did not function as a shuttle-like electron carrier between the menaquinol:cytochrome c oxidoreductase and cytochrome c(z). It was also shown that the rereduction rate of cytochrome c(z) by cytochrome c-555 was as high as that by the menaquinol:cytochrome c oxidoreductase. The two electron-transfer pathways linked to sulfur metabolisms seem to function independently to donate electrons to the reaction center.     

Generation of polymerase chain reaction-specific probes for library screening using single degenerate primers

Degenerate oligonucleotide primers were made to peptide sequences from hydroxylamine oxidoreductase (HAO) from Nitrosomonas europaea. The primers were used singly in PCR reactions to amplify portions of the gene for HAO from genomic DNA. Southern hybridizations using fragments amplified with each primer showed that they labeled the same genomic DNA fragments. The PCR-amplified fragments were successfully used to screen a gene library for clones containing the HAO gene. The method of isolating genes by PCR with single primers has general utility.     

Complexity in the redox titration of the dihaem cytochrome c4

Redox titration of the dihaem, two domain cytochromes c4 from Pseudomonas aeruginosa, Pseudomonas stutzeri and Azotobacter vinelandii showed complex behaviour indicative of the presence of two redox components. In the case of the P. stutzeri cytochrome c4, two spectroscopically distinct components were present during the redox titration. In contrast, cytochrome c-554(548) from a halophilic Paracoccus species is a stable dimer of a monohaem cytochrome which shows close homology to cytochrome c4, but does not show complexity in its redox titration. The presence of chemically distinct haem environments or anti-cooperative interactions between identical haem groups are two possible explanations for the redox complexity of cytochrome c4. The simple redox titration of cytochrome c-554(548) shows that haems situated relatively close together need not interact, but direct cleavage, separation and study of the domains will be necessary to decide whether they do or do not interact in the case of cytochrome c4.     

Inhibition of Tn554 transposition: Deletion analysis

Tn554, a transposon in Staphylococcus aureus that specifies resistance to erythromycin and spectinomycin, exhibits a high preference for a single chromosomal insertion site. If this site is already occupied by a copy of Tn554, the transposition of a second element is inhibited 100- to 1000-fold. This report defines the locus of the inhibitory activity and presents both a functional and a restriction map of Tn554. Fragments containing parts of Tn554 were cloned on an autonomously replicating plasmid. Those clones containing the "left" end of Tn554 strongly inhibited the transposition of an incoming, intact copy of Tn554. Analysis of deleted derivatives of these clones defined a locus tnpI, which is both necessary and sufficient for transpositional inhibition. This locus consists of the terminal 89 bp of the "left" end of Tn554. It is suggested that this terminal sequence acts to titrate a factor required for transposition.     

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.