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PubMed1987

Isolation and characterization of ubiquinol oxidase complexes from Paracoccus denitrificans cells cultured under various limiting growth conditions in the chemostat.

To obtain more information about the composition of the respiratory chain under different growth conditions and about the regulation of electron-transfer to several oxidases and reductases, ubiquinol oxidase complexes were partially purified from membranes of Paracoccus denitrificans cells grown in carbon-source-limited aerobic, nitrate-limited anaerobic and oxygen-limited chemostat cultures. The isolated enzymes consisted of cytochromes bc1, c552 and aa3. In comparison with the aerobic ubiquinol oxidase complex, the oxygen- and nitrate-limited ones contained, respectively, less and far less of the cytochrome aa3 subunits and the anaerobic complex also contained lower amounts of cytochrome c552. In addition, extra haem-containing polypeptides were present with apparent Mr of 14,000, 30,000 and 45,000, the former one only in the anaerobic and the latter two in both the anaerobic and oxygen-limited preparations. This is the first report describing four different membrane-bound c-type cytochromes. The potentiometric and spectral characteristics of the redox components in membrane particles and isolated ubiquinol oxidase fractions were determined by combined potentiometric analysis and spectrum deconvolution. Membranes of nitrate- and oxygen-limited cells contained extra high-potential cytochrome b in comparison with the membranes of aerobically grown cells. No difference was detected between the three isolated ubiquinol oxidase complexes. Aberrances with already published values of redox potentials are discussed.

Aerobiosis↗

PubMed1987

Subfractionation and characterization of soluble c-type cytochromes from Paracoccus denitrificans cultured under various limiting conditions in the chemostat.

Soluble c-type cytochromes were partially purified from Paracoccus denitrificans cells grown in succinate- and methanol-limited aerobic, nitrate-limited anaerobic and oxygen-limited chemostat cultures. Five c types could be distinguished with the following apparent molecular masses, absorption maxima and midpoint potentials. (a) 9.2 kDa, 549 nm and +190 mV; (b) 14 kDa, 549 nm and +227 mV; (c) 22 kDa, 552 nm and +190 mV; (d) 30 kDa, 552.7 nm and +160 mV; (e) 45 kDa, a dihaem: 555 nm, +128 mV and 551 nm, -163 mV. The 14-kDa polypeptide was present under all growth conditions examined and most probably is the already well characterized cytochrome c550. In methanol-limited grown cells three additional cytochromes were found, the 9.2-kDa, 22-kDa and 30-kDa ones. Under oxygen-limited conditions the 45-kDa and under anaerobic growth conditions small quantities of the 30-kDa and 45-kDa cytochromes c were present. Based on the apparent molecular masses the 14-kDa, 22-kDa, 30-kDa and 45-kDa cytochromes may also be present in membrane-fractions.

Aerobiosis↗

PubMed1988

Respiratory nitrate reductase from Paracoccus denitrificans. Evidence for two b-type haems in the gamma subunit and properties of a water-soluble active enzyme containing alpha and beta subunits.

1. The b-type haem centres of the three (alpha, beta and gamma) subunit nitrate reductase from Paracoccus denitrificans have been analysed by redox potentiometry. Two components were identified with mid-point potentials +95 mV and +210 mV. 2. Washing, in the absence of Mg2+ ions, of cytoplasmic membrane vesicles from P. denitrificans promoted selective release of nitrate reductase activity. The released enzyme was purified by chromatography and shown to contain alpha and beta, but not gamma polypeptides. A haem spectrum was absent, consistent with the lack of the gamma subunit. The alpha and beta polypeptides of the water-soluble nitrate reductase had molecular masses that were identical to those of the detergent-purified enzyme and also of the nitrate reductase in cytoplasmic membranes. This observation, together with the failure of protease inhibitors to prevent release from the membrane, indicates that the release is not related to limited proteolysis of the alpha and/or beta polypeptides. The relative molecular mass of the water-soluble alpha beta enzyme was estimated to be approximately 200,000. 3. The water-soluble nitrate reductase was released from intact inverted cytoplasmic membrane vesicles as judged by loss of NADH-NO3- reductase activity and retention by the vesicles after washing of uncoupler-sensitive NADH-oxidase activity. These observations show that alpha and beta polypeptides, and therefore the active site for nitrate reduction, are located on the cytoplasmic side of the membrane. 4. Attempts to reverse the nitrate reductase activity of the enzyme, using nitrate as reductant plus ferricyanide or chlorate as tested oxidants, were unsuccessful. The implications for the mechanism of the enzyme are discussed.

Cytoplasm↗

PubMed1989

The energy-conserving nitric-oxide-reductase system in Paracoccus denitrificans. Distinction from the nitrite reductase that catalyses synthesis of nitric oxide and evidence from trapping experiments for nitric oxide as a free intermediate during denitrification.

1. A Clark-type electrode that responds to nitric oxide has been used to show that cytoplasmic membrane vesicles of Paracoccus denitrificans have a nitric-oxide reductase activity. Nitrous oxide is the reaction product. NADH, succinate or isoascorbate plus 2,3,5,6-tetramethyl-1,4-phenylene diamine can act as reductants. The NADH-dependent activity is resistant to freezing of the vesicles and thus the NADH:nitric-oxide oxidoreductase activity of stored frozen vesicles provides a method for calibrating the electrode by titration of dissolved nitric oxide with NADH. The periplasmic nitrite reductase and nitrous-oxide reductase enzymes are absent from the vesicles which indicates that nitric-oxide reductase is a discrete enzyme associated with the denitrification process. This conclusion was supported by the finding that nitric-oxide reductase activity was absent from both membranes prepared from aerobically grown P. denitrificans and bovine heart submitochondrial particles. 2. The NADH: nitric-oxide oxidoreductase activity was inhibited by concentrations of antimycin or myxothiazol that were just sufficient to inhibit the cytochrome bc1 complex of the ubiquinol--cytochrome-c oxidoreductase. The activity was deduced to be proton translocating by the observations of: (a) up to 3.5-fold stimulation upon addition of an uncoupler; and (b) ATP synthesis with a P:2e ratio of 0.75. 3. Nitrite reductase of cytochrome cd1 type was highly purified from P. denitrificans in a new, high-yield, rapid two- or three-step procedure. This enzyme catalysed stoichiometric synthesis of nitric oxide. This observation, taken together with the finding that the maximum rate of NADH:nitric-oxide oxidoreductase activity catalysed by the vesicles was comparable with that of NADH:nitrate-oxidoreductase, is consistent with a role for nitric-oxide reductase in the physiological conversion of nitrate or nitrite to dinitrogen gas. 4. Intact cells of P. denitrificans also reduced nitric oxide in an antimycin- or myxothiazol-sensitive manner. However, nitric oxide was not detected by the electrode during the reduction of nitrate. Nitric-oxide synthesis from nitrate could be detected with cells in the presence of very low concentrations of Triton X-100 which selectively inhibits nitric-oxide reductase activity. 5. Nitric oxide was detected as an intermediate in denitrification by including haemoglobin with an anaerobic suspension of cells that was reducing nitrate. The characteristic spectrum of the nitric oxide derivative of haemoglobin was observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Cytoplasm↗

PubMed1993

Proton slippage in cytochrome c oxidase of Paracoccus denitrificans. Membrane-potential measurements with the two-subunit and three-subunit enzyme.

Isolated cytochrome c oxidase from Paracoccus denitrificans, containing either two or three subunits, was reconstituted into liposomes and the membrane potential was measured at different rates of respiration using a triphenylmethylphosponium bromide electrode. Both enzymes revealed a non-linear increase of the membrane potential with increasing respiratory rates. The ratios of the respiratory rates of the two proton pumps decreased with increasing membrane potential, suggesting slippage of proton pumping, as has been shown before with two cytochrome c oxidases from bovine heart, differing in H+/e- stoichiometries due to chemical modification [Steverding, D. & Kadenbach, B. (1991) J. Biol. Chem. 266, 8097-8101]. The data suggest that slippage of proton pumping represents an intrinsic property of cytochrome c oxidase associated with the two catalytic subunits, I and II.

Electron Transport Complex IV↗

PubMed1993

Mutants of Methylobacterium extorquens and Paracoccus denitrificans deficient in c-type cytochrome biogenesis synthesise the methylamine-dehydrogenase polypeptides but cannot assemble the tryptophan-tryptophylquinone group.

Five mutants of Methylobacterium extorquens and four mutants of Paracoccus denitrificans that have a general defect in c-type cytochrome synthesis also failed to assemble an active methylamine dehydrogenase. In all cases methanol dehydrogenase, another periplasmic enzyme, was fully active. All nine mutant strains accumulated both the heavy and light subunits of methylamine dehydrogenase to essentially wild-type levels. In all nine mutants, the heavy-subunit and light-subunit polypeptides were proteolytically processed, suggesting that translocation to the periplasm had occurred; in the case of the P. denitrificans mutants, a periplasmic location for the heavy and light subunits was confirmed experimentally. While specific quinone staining of the methylamine dehydrogenase light subunit in wild-type M. extorquens and P. denitrificans strains could readily be demonstrated, the light subunit polypeptides accumulated by the mutants did not quinone stain, indicating that the methylamine dehydrogenase prosthetic group, tryptophan tryptophylquinone, is not assembled in the absence of functional c-type cytochromes.

Benzoquinones↗

PubMed1994

Expression of the mau genes involved in methylamine metabolism in Paracoccus denitrificans is under control of a LysR-type transcriptional activator.

Expression of methylamine dehydrogenase in Paracoccus denitrificans and its concomitant ability to grow on methylamine is regulated by a substrate-induction mechanism as well as by a catabolite-repression-like mechanism. Methylamine dehydrogenase is synthesized in cells growing on either methylamine or ethylamine, but not during growth on succinate, methanol or choline as sole sources of carbon and energy. The synthesis of methylamine dehydrogenase is repressed when succinate is added to the growth medium in addition to methylamine. Repression is not observed when the growth medium contains methylamine and either choline or methanol. Induction of the mau genes encoding methylamine dehydrogenase is under control of the mauR gene. This regulatory gene is located directly in front of, but with the transcription direction opposite to that of, the structural genes in the mau cluster. The mauR gene encodes a LysR-type transcriptional activator. Inactivation of the gene results in loss of the ability to synthesize methylamine dehydrogenase and amicyanin, and loss of the ability to grow on methylamine. The mutation is completely restored when the mauR gene is supplied in trans. The first gene of the cluster of mau genes that is under control of MauR is mauF, which encodes a putative membrane-embedded protein. Inactivation of the gene results in the inability of cells to grow on methylamine. Downstream from mauF and in the same transcription direction, mauB is located. This gene encodes the large subunit of methylamine dehydrogenase.

Amino Acid Sequence↗

PubMed1995

The oxidation of methylamine in Paracoccus denitrificans.

The in vivo oxidation of methylamine has been studied in Paracoccus denitrificans. Four components are involved in the electron transfer from methylamine to oxygen; methylamine dehydrogenase (MADH), amicyanin, cytochrome c and cytochrome-c oxidase. In P. denitrificans, MADH and its electron acceptor amicyanin are indispensable for growth on methylamine. In the present study, site-directed mutants have been used to demonstrate participation of cytochrome c550 and the aa3-type cytochrome-c oxidase. Moreover, evidence is provided for the operation of alternative routes, branching from amicyanin, in which at least cytochrome c1 and the cbb3-type cytochrome-c oxidase are involved.

Methylamines↗

PubMed1995

Perturbation of the CuA site in cytochrome-c oxidase of Paracoccus denitrificans by replacement of Met227 with isoleucine.

Subunit II of cytochrome-c oxidase contains a redox centre, CuA, with unusual spectroscopic properties; this site consists of two copper atoms and acts as the entry point for electrons from cytochrome c. We have constructed a site-directed mutant of cytochrome-c oxidase from Paracoccus denitrificans in which the CuA site has been disturbed by replacement of Met227 with isoleucine. The purified, fully assembled enzyme complex has been investigated with various techniques including metal analysis, EPR and visible spectroscopies, steady-state and fast kinetics. The stoichiometry of the metals in the enzyme remains unchanged but a clear perturbation of the CuA site can be observed in the EPR and near-infrared optical spectra. It is concluded that in the mutant CuA is still binuclear but that the two nuclei are no longer equivalent, converting the delocalized [Cu(1.5)....Cu(1.5)] centre of the wild type into a localized [Cu(I)....Cu(II)] system. Changes in the overall kinetics of the mutant are correlated with a diminished electron transfer rate between CuA and heme alpha.

Copper↗

PubMed1995

The affinity and specificity of Ca(2+)-binding sites of cytochrome-c peroxidase from Paracoccus denitrificans.

The binding of Ca2+ to the dihaem cytochrome-c peroxidase from Paracoccus denitrificans was analysed by following perturbations in the visible and 1H-NMR spectra of both haem groups. The enzyme contains at least two types of Ca(2+)-binding site. Site I is occupied in the isolated enzyme, binds Ca2+ with a redox-state-independent Kd of 1.2 microM and accommodates neither Mg2+ nor Mn2+. Site II is unoccupied in dilute solutions of the isolated oxidised enzyme and binds Ca2+ cooperatively with a Kd of 0.52 mM. In the mixed valence form, the binding affinity increases to resemble that of site I. The cooperativity was shown by -Ca2+ binding to site II, the titration of haem methyl 1H-NMR resonances, and a half-of-sites effect observed for modification of an essential histidine with diethylpyrocarbonate. These are all consistent with site II being situated at the interface between two monomers of a dimeric enzyme. Thus the equilibrium of binding to site II is a reflection of the equilibrium for dimerisation and conditions which shift that equilibrium towards the dimer, such as increased ionic strength or high protein concentration, also increase Ca2+ affinity. Binding of Ca2+ to site II is required for formation of the active high spin state at the peroxidatic haem.

Ascorbic Acid↗

PubMed1995

Mutational analysis of mau genes involved in methylamine metabolism in Paracoccus denitrificans.

A chromosomal fragment containing DNA downstream from mauC was isolated from Paracoccus denitrificans. Sequence analysis of this fragment revealed the presence of four open reading frames, all transcribed in the same direction. The products of the putative genes were found to be highly similar to MauJ, MauG, MauM and MauN of Methylobacterium extorquens AM1. Using these four mau genes, 11 mau genes have been cloned from P. denitrificans to date. The gene order is mauRFBEDACJGMN, which is similar to that in M. extorquens AM1. mauL, present in M. extorquens AM1, seems to be absent in P. denitrificans. MauJ is predicted to be a cytoplasmic protein, and MauG a periplasmic protein. The latter protein contains two putative heme-binding sites, and has some sequence resemblance to the cytochrome c peroxidase from Pseudomonas aeruginosa. MauM is also predicted to be located in the periplasm, but MauN appears to be membrane associated. Both resemble ferredoxin-like proteins and contain four and two motifs, respectively, characteristic for [4Fe-4S] clusters. Inactivation of mauA, mauJ, mauG, mauM and mauN was carried out by introduction of unmarked mutations in the chromosomal copies of these genes. mauA and mauG mutant strains were unable to grow on methylamine. The mauJ mutant strain had an impaired growth rate and showed a lower dye-linked methylamine dehydrogenase (MADH) activity than the parent strain. Mutations in mauM and mauN had no effect on methylamine metabolism. The mauA mutant strain specifically lacked the beta subunit of MADH, but the alpha subunit and amicyanin, the natural electron acceptors of MADH, were still produced. The mauG mutant strain synthesized the alpha and beta subunits of MADH as well as amicyanin. However, no dye-linked MADH activity was found in this mutant strain. In addition, as the wild-type enzyme displays a characteristic fluorescence emission spectrum upon addition of methylamine, this property was lost in the mauG mutant strain. These results clearly show that MauG is essential for the maturation of the beta subunit of MADH, presumably via a step in the biosynthesis of tryptophan tryptophylquinone, the cofactor of MADH. The mau gene cluster mauRFBEDACJGMN was cloned on the broad-host vector pEG400. Transfer of this construct to mutant strains which were unable to grow on methylamine fully restored their ability to grow on this compound. A similar result was achieved for the closely related bacterium Thiosphaera pantotropha, which is unable to utilize methylamine as the sole sources of carbon and energy.

Amino Acid Sequence↗

PubMed1995

Spectroscopic and mutagenesis studies on the CuA centre from the cytochrome-c oxidase complex of Paracoccus denitrificans.

Cytochrome-c oxidase contains an unusual copper centre (CuA) located in subunit II. This centre mediates one-electron transfer from cytochrome c to low-spin heme a. Recent spectroscopic and biochemical studies have shown that this centre is a valence delocalised dinuclear [Cu(+1.5)-Cu(+1.5)] centre. We have measured the absorption, EPR and variable-temperature magnetic circular dichroism spectra of the CuA-binding domain isolated from Paracoccus denitrificans cytochrome aa3. The EPR spectrum showed the following signals: gparallel = 2.18; gperpendicular = 2.03. gparallel exhibited a seven-line hyperfine splitting pattern, with an intensity ratio showing that the single unpaired electron interacted equally with two copper nuclei. The magnetic circular dichroism spectrum was identical to those from CuA in bovine heart cytochrome-c oxidase and centre A of nitrous-oxide reductase, showing the close structural similarity between the three centres. To identify the ligands of CuA, all the conserved putative ligands in the P. denitrificans CuA domain were substituted. Only five residues, Cys244, Cys248, His209, His252, and Met255, were required for correct assembly of the CuA centre. Replacement of Met255 caused protein misfolding. Hence, methionine may have a structural role for the folding of the protein rather than being a CuA ligand. Given that both copper ions must have identical coordination geometries, the number of possible structures is limited. Two models are proposed: one involves the thiolate side-chains of Cys244 and Cys248 bridging a pair of copper ions with one histidine coordinating each copper ion, and the other has terminal ligation of each copper ion by one cysteine and one histidine residue. In both models, the metal-metal distance can be sufficiently short to permit direct d-orbital overlap of the copper ions. The magnetic circular dichroism transitions at 475 nm and 525 nm are assigned to thiolate-to-copper charge-transfer processes polarised perpendicular to one another, although the magnetic circular dichroism intensities show that the excited states were heavily mixed with copper d-orbitals. These intensities can be interpreted in the thiolate bridged model in terms of transitions within a Cu2(SR)2 rhomb. In the model involving terminal cysteine ligation, exciton coupling of two thiolate-to-copper charge-transfer transitions of similar energy, polarised along the Cu-S bonds, would contribute two transitions perpendicular to one another. This requires that the cysteine ligands have a cis orientation relative to one another.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1997

Molecular cloning and functional characterization of the Paracoccus denitrificans porin.

Bacterial porins facilitate the passive uptake of small solutes across the outer membrane of the cell. The channel properties and the primary structure of the porin from Paracoccus denitrificans were investigated. As judged from single-channel conductance experiments, this porin forms trimeric pores that show no ion selectivity in potassium chloride solution, which indicates that the charges within or near the channel are balanced. Based on peptide fragment sequence, the gene porG, which codes for this general pore protein, was cloned and analyzed. Its primary translation product contains a 20-residue signal sequence, followed by the 295 amino acids of the mature protein with a molecular mass of 31.9 kDa. Sequence alignments with porins from Rhodopseudomonas blastica and Rhodobacter capsulatus and secondary structure predictions suggest a typical rigid barrel structure consisting of 16 antiparallel beta-strands.

Amino Acid Sequence↗

PubMed1997

Expression studies on the ba3 quinol oxidase from Paracoccus denitrificans. A bb3 variant is enzymatically inactive.

Expression of the quinol oxidase from Paracoccus denitrificans has been examined using a polyclonal antibody directed against subunit II and a promoter probe vector carrying the promoter region of the qox operon. Under aerobic conditions nitrate and nitrite act as specific inducers of the expression. To obtain an enzymatically competent quinol oxidase complex, an intact ctaB gene is required, which constitutes part of the cta operon coding for the aa3 cytochrome c oxidase of P. denitrificans. Deletion of ctaB leads to a change in heme composition of the quinol oxidase with heme b replacing the high-spin heme a of the binuclear center, causing loss of electron transport activity.

Antibodies↗

PubMed1995

Analysis of beta-ketothiolase and acetoacetyl-CoA reductase genes of a methylotrophic bacterium, Paracoccus denitrificans, and their expression in Escherichia coli.

The beta-ketothiolase gene (phaA) and acetoacetyl-CoA reductase gene (phaB) were isolated from Paracoccus denitrificans. Nucleotide sequence analysis showed that they encoded proteins of 391 amino acids with a molecular mass of 40,744 Da and of 242 amino acids with a molecular mass of 25,614 Da, respectively. The predicted gene products exhibited high amino acid identities with those from other bacteria: 64.4-74.0% for the phaA gene product and 47.6-80.6% for the phaB gene product, respectively. Both genes were co-transcribed in a recombinant Escherichia coli. In addition, promoter activity was detected upstream of the phaA gene. Hence, the two genes are organized as an operon, phaA-phaB, in P. denitrificans. NADH was preferred to NADPH as a cofactor of acetoacetyl-CoA reductase.

Acetyl-CoA C-Acyltransferase↗

PubMed1996

Is the Paracoccus halodenitrificans ATPase a chimeric enzyme?

Membranes from Paracoccus halodenitrificans contain an ATPase that is most active in the absence of NaCl. The most unusual characteristic of the enzyme is its pattern of sensitivity to various inhibitors. Azide and rhodamine 6G, inhibitors of F1F0-ATPases, inhibit ATP hydrolysis as do bafilomycin A1, concanamycin A (folimycin), N-ethylmaleimide, and p-chloromercuriphenylsulfonate which are inhibitors of vacuolar ATPases. This indiscriminate sensitivity suggests that this ATPase may be a hybrid and that caution should be exercised when using inhibition as a diagnostic for distinguishing between F1F0-ATPases and vacuolar ATPases.

Adenosine Triphosphatases↗

PubMed1996

Oxygen increases the steady-state level of nitrate in denitrifying cells of Paracoccus denitrificans.

The levels of nitrate in denitrifying cells of Paracoccus denitrificans were determined by centrifugation through silicone oil into phosphoric acid and ion-exchange HPLC analysis of the cell lysates, using [14C]sucrose to correct for the trapped external medium. Introduction of oxygen brought about a significant upward shift in the intracellular nitrate concentration. This result calls into question the current thinking that oxygen blocks nitrate respiration primarily due to the inhibition of nitrate transport into the cell.

Biological Transport↗

PubMed1997

Cloning and characterization of the recA of Paracoccus denitrificans and construction of a recA-deficient mutant.

The recA gene of Paracoccus denitrificans has been isolated from a genomic library by hybridization with the Rhodobacter sphaeroides recA gene. Its complete nucleotide sequence consists of 1071 bp encoding a polypeptide of 356 amino acids. Nucleotide sequence analysis of the P. denitrificans recA gene revealed the closest identities with the R. sphaeroides and the Rhodobacter capsulatus recA genes. Nevertheless, and surprisingly, recA genes of these two phototrophic bacteria are not DNA damage-inducible when introduced into P. denitrificans cells, whereas recA genes of both P. denitrificans and Rhizobium etli are. These results suggest that the promoters of P. denitrificans and R. etli recA genes have a similar regulatory sequence. A recA-defective mutant of P. denitrificans has also been constructed by replacement of the active recA gene by an in vitro inactivated gene copy.

Cloning, Molecular↗