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PubMed1995

Reversed electron transfer through the bc1 complex enables a cytochrome c oxidase mutant (delta aa3/cbb3) of Paracoccus denitrificans to grow on methylamine.

In Paracoccus denitrificans four classes of redox proteins are involved in the electron transfer from methylamine to oxygen:methylamine dehydrogenase (MADH), amicyanin, cytochrome c and cytochrome c oxidase. MADH and its electron acceptor amicyanin are indispensable for growth on methylamine. At least three different cytochromes c and two types of cytochrome c oxidase, cytochromes aa3 and cbb3, have previously been proposed to participate in the electron transfer pathways from methylamine to oxygen. In this study, participation of both cytochrome c oxidases and of the quinol oxidase (cytochrome bb3) has indeed been confirmed by analysis of a series of oxidase mutants. Interestingly, a P. denitrificans cytochrome c oxidase mutant (delta aa3/cbb3) retains the capacity to oxidise methylamine. It is demonstrated that the oxidation of the cytochrome c pool in this mutant does not proceed via an alternative cytochrome c oxidase, but rather via an 'uphill' electron transfer through the bc1 complex to ubiquinone, driven by the membrane potential. The subsequent oxidation of ubiquinol proceeds via the only remaining terminal oxidase, the bb3-type quinol oxidase.

Culture Media↗

PubMed1996

Carboxyl group protonation upon reduction of the Paracoccus denitrificans cytochrome c oxidase: direct evidence by FTIR spectroscopy.

The redox reactions of the cytochrome c oxidase from Paracoccus denitrificans were investigated in a thin-layer cell designed for the combination of electrochemistry under anaerobic conditions with UV/VIS and IR spectroscopy. Quantitative and reversible electrochemical reactions were obtained at a surface-modified electrode for all cofactors as indicated by the optical signals in the 400-700 nm range. Fourier transform infrared (FTIR) difference spectra of reduction and oxidation (reduced-minus-oxidized and oxidized-minus-reduced, respectively) obtained in the 1800-1000 cm(-1) range reveal highly structured band features with major contributions in the amide I (1620-1680 cm(-1)) and amide II (1580-1520 cm(-1)) range which indicate structural rearrangements in the cofactor vicinity. However, the small amplitude of the IR difference signals indicates that these conformational changes are small and affect only individual peptide groups. In the spectral region above 1700 cm(-1), a positive peak in the reduced state (1733 cm(-1)) and negative peak in the oxidized st ate (1745 cm(-1)) are characteristic for the formation and decay of a COOH mode upon reduction. The most obvious interpretation of this difference signal is proton uptake by one Asp or Glu side chain carboxyl group in the reduced state and deprotonation of another Asp or Glu residue. Moreover, both residues could well be coupled as a donor-acceptor pair in the proton transfer chain. An alternative interpretation is in terms of a protonated carboxyl group which shifts to a different environment in the reduced state. The relevance of this first direct observation of protein protonation changes in the cytochrome c oxidase for vectorial proton transfer and the catalytic reaction is discussed.

Electron Transport Complex IV↗

PubMed1996

The purification of ammonia monooxygenase from Paracoccus denitrificans.

The heterotrophic nitrifier Paracoccus denitrificans expresses a membrane-associated ammonia monooxygenase. The active enzyme has been solubilized in the detergent dodecyl-beta-D-maltoside and purified by standard chromatographic techniques. This is the first purification of an ammonia monooxygenase. The enzyme consists of two subunits with molecular masses of 38 and 46 kDa. The purified enzyme is a quinol oxidase, is inhibited by light and a variety of chelating agents and is activated by cupric ions. These properties indicate that this enzyme has similarities to a family of enzymes including the ammonia monooxygenase from Nitrosomonas europaea and the particulate methane monooxygenase from Methylococcus capsulatus (Bath).

Oxidoreductases↗

PubMed1996

Genetic inactivation of the H(+)-translocating NADH:ubiquinone oxidoreductase of Paracoccus denitrificans is facilitated by insertion of the ndh gene from Escherichia coli.

The H(+)-translocating NADH:ubiquinone oxidoreductase (NDH1) is probably an obligatory enzyme in Paracoccus denitrificans and disruption of its genes may be lethal to this organism. In order to overcome this problem and delete the nqo8 and nqo9 genes of NDH1, it was necessary to render the enzyme non-essential. This was achieved by constructing a deletion plasmid in which most of the coding regions of nqo8 and nqo9 were replaced by the ndh gene of Escherichia coli that encodes an alternative NADH:ubiquinone oxidoreductase (NDH2), and a kanamycin resistance gene. Subsequent homologous recombination gave rise to a mutant the membranes of which catalyzed rotenone-insensitive NADH oxidation, but which did not oxidize deamino-NADH. Hence, this mutant expressed active and membrane-bound NDH2, and lacked NDH1 activity.

Amino Acid Sequence↗

PubMed1990

Rapid purification of cytochrome c oxidase from Paracoccus denitrificans.

Two methods are described for the purification of cytochrome c oxidase from Triton X-100 extracts of the periplasma membrane of Paracoccus denitrificans. The first is a large-scale procedure for the preparation of 100-250 nmol of cytochrome c oxidase (10-20 mg) in 1 week. The second is a rapid procedure for isolating up to 25 nmol in 2-3 days. Owing to the high yields given by fast protein liquid chromatography (FPLC) on Mono Q columns, the overall yield is about 20%, whereas the yield in many other previously published procedures does not exceed 10%. The use of FPLC on Mono Q also offers a considerable saving of time.

Cell Membrane↗

PubMed1986

Preliminary X-ray crystallographic study of amicyanin from Paracoccus denitrificans.

Single crystals have been prepared of Paracoccus denitrificans amicyanin, a blue copper protein that serves as an electron acceptor for methylamine dehydrogenase. The crystals belong to the monoclinic space group P2(1), and have unit cell parameters a = 20.90 A, b = 56.61 A, c = 27.55 A and beta = 96.41. There is one molecule in the asymmetric unit. The crystals diffract to beyond 1.5 A resolution.

Bacterial Proteins↗

PubMed1983

Production, isolation and characterization of monoclonal antibodies to cytochromes c of beef heart and Paracoccus denitrificans.

Hybridoma cell lines secreting monoclonal antibodies which bind beef heart cytochrome c or Paracoccus denitrificans cytochrome c have been produced using spleen cells from BALB/c mice immunized with cytochrome c. Immunization was performed with either the native cytochrome c, succinylated hemocyanin-conjugated cytochrome c, or beef heart cytochrome c polymerized with glutaraldehyde. Of 10 such fusions, the hybridization frequency ranged from 0 to 42%. The cell fusion efficiency, the possible factors involved in the cell fusion efficiency and the frequency of antibody producing hybridomas are described. The percentage of hybridomas positive for anti-cytochrome c antibody production as screened for by radioimmunoassay or ELISA was 2%. Of the antibodies from 12 hybridoma cell lines which resulted from 10 fusions, three were specific to beef heart cytochrome c, another three were specific to P. denitrificans cytochrome c, and the remainder reacted with both cytochromes c. These groups of monoclonal antibodies react to different sets of sites on these two cytochromes c. The monoclonal antibodies from ten representative clones have been isolated and characterized by different methods.

Animals↗

PubMedDate not supplied

Cloning of Paracoccus cytochrome c oxidase subunit II.

Cytochrome c oxidase from Paracoccus denitrificans is composed of two subunits, yet is active in both electron transport and proton translocation. A cloning approach and immunologic screening protocol is described for the isolation of the subunit II gene expressed in E. coli. DNA sequencing should establish the extent of homology to eukaryotic oxidase.

Cloning, Molecular↗

PubMed1982

Isolation and characterization of a protein with cyanide-sensitive superoxide dismutase activity from the prokaryote, Paracoccus denitrificans.

1. A protein with cyanide-sensitive superoxide dismutase activity was isolated from the prokaryote Paracoccus denitrificans. 2. This enzyme, present in low amount in the cell, represented not more than 10% of the total cellular superoxide dismutase activity. It was obtained in a form which was 20-40-times less active than the main superoxide dismutase of P. denitrificans which is a manganese-containing enzyme. 3. It was a soluble monomeric enzyme, highly negatively charged (pI = 4.8), with an apparent molecular weight of 33,000. 4. Cyanide sensitivity was observed by NMR assay, enzyme assay and by staining the protein for superoxide dismutase activity on polyacrylamide electrophoretogram. KCN was shown to be a competitive inhibitor of this dismutase, with an inhibitor constant of 0.15 mM. 5. From the amino acid analysis, S delta Q values lower than 100 were obtained with copper-containing proteins such as the subunit II of cytochrome oxidase from P. denitrificans (69), the azurin from P. denitrificans (77), the bacteriocuprein from Photobacter leiognathi (71); with iron and manganese superoxide dismutases (40-88), and with some eukaryotic copper/zinc dismutases of fish origin (55-82).

Amino Acids↗

PubMed1992

Cofactor-directed inactivation by nucleophilic amines of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans.

Phenylhydrazine, semicarbazide, aminoguanidine, hydrazine, and hydroxylamine each irreversibly inactivated methylamine dehydrogenase from Paracoccus denitrificans and caused changes in the absorbance spectrum of the protein-bound tryptophan tryptophylquinone [TTQ] prosthetic group. Different spectral perturbations were observed on reaction with each of these inactivators. In each case a stoichiometry of 2 mol per mol of enzyme (1:1 per cofactor) was required to observe complete modification of the absorbance spectrum. Identical changes were observed in the presence and absence of oxygen. The reactions of hydrazine and hydroxylamine were very rapid, with stoichiometric inactivation occurring in less than 30 s. Inactivation by phenylhydrazine and semicarbazide exhibited apparent bimolecular kinetics and second order rate constants for inactivation, respectively, of 25 min-1 mM-1 and 39 min-1 mM-1. In contrast, inactivation by aminoguanidine exhibited saturation behavior and kinetic parameters of KI = 2.5 mM and kinact = 0.5 min-1 were obtained. Ammonium salts did not inactivate the enzyme, but were reversible competitive inhibitors with respect to methylamine. A Ki of 20 mM was obtained for ammonium chloride. A mechanism for the reactions of these compounds with the TTQ cofactor of methylamine dehydrogenase is proposed, and the relationship of these data to the mechanisms of interaction of these compounds with o-quinones and other quinoproteins which possess TTQ and other quinone cofactors is discussed.

Amines↗

PubMed1989

Assimilation of methylamine by Paracoccus denitrificans involves formaldehyde transport by a specific carrier.

Assimilation of methylamine by Paracoccus denitrificans involves the following enzymes: a periplasmic methylamine dehydrogenase, a formaldehyde transport system, cytoplasmic formaldehyde and formate dehydrogenase. Formaldehyde transport follows saturation kinetics with a high substrate affinity (Km = 7 microM), and is severely inhibited by iodoacetate, cyanide and p-trifluoromethoxy carbonylcyanide phenylhydrazone. Expression of the formaldehyde carrier is regulated by the carbon source.

Adenosine Triphosphate↗

PubMed1992

An FNR-dependent promoter from Escherichia coli is active and anaerobically inducible in Paracoccus denitrificans.

A transcriptional fusion of a synthetic FNR-dependent promoter derived from Escherichia coli has been introduced into Paracoccus denitrificans on a broad-host-range plasmid. The patterns of expression of beta-galactosidase from this fusion and from a control which is not regulated by FNR have been studied. The results indicate that P. denitrificans expresses a transcriptional regulator which has a very similar DNA-binding specificity to FNR, and responds to a similar physiological signal.

Anaerobiosis↗

PubMed1992

Factors affecting the stability of methanol dehydrogenase from Paracoccus denitrificans.

Methanol dehydrogenase from Paracoccus denitrificans was purified to homogeneity in two steps from the periplasmic fraction of methanol-grown cells. The enzyme was composed of subunits of M(r) 67,000 and 12,000, and non-covalently bound pyrroloquinoline quinone. It exhibited a pH optimum at pH values of 9.0 and above. It was not stable at pH greater than 9.0, but exhibited little loss of activity after prolonged incubation at pH values as low as 4.5. Methyl dehydrogenase was relatively stable to thermal denaturation. The thermal stability was enhanced by the presence of Ca2+ and diminished by the presence of EDTA. These data suggest a structural role for Ca2+ in this enzyme, similar to what has been observed with quinoprotein glucose and ethanol dehydrogenases.

Alcohol Oxidoreductases↗

PubMed1993

Synthesis of the Rhodopseudomonas viridis holo-cytochrome c2 in Paracoccus denitrificans.

The gene encoding the Rhodopseudomonas viridis cytochrome c2 (cycA) has been introduced on a broad host range vector into Paracoccus denitrificans, leading to high-level expression of the holo-cytochrome with the heme moiety covalently attached to the apoprotein. The cytochrome was demonstrated to reside in the periplasmic space of the host cell. In contrast to R. viridis, aerobic rather than anaerobic growth conditions led to higher production levels of the holo-cytochrome in P. denitrificans. This heterologous expression system provides a suitable genetic background for the functional expression and mutagenesis of polypeptides involved in bacterial photosynthesis, offering the possibility of detailed structural and functional investigation.

Aerobiosis↗

PubMed2006

Requirement of medium ADP for the steady-state hydrolysis of ATP by the proton-translocating Paracoccus denitrificans Fo.F1-ATP synthase.

Fo.F1-ATP synthase in inside-out coupled vesicles derived from Paracoccus denitrificans catalyzes Pi-dependent proton-translocating ATPase reaction if exposed to prior energization that relieves ADP.Mg2+ -induced inhibition (Zharova, T.V. and Vinogradov, A.D. (2004) J. Biol. Chem.,279, 12319-12324). Here we present evidence that the presence of medium ADP is required for the steady-state energetically self-sustained coupled ATP hydrolysis. The initial rapid ATPase activity is declined to a certain level if the reaction proceeds in the presence of the ADP-consuming, ATP-regenerating system (pyruvate kinase/phosphoenol pyruvate). The rate and extent of the enzyme de-activation are inversely proportional to the steady-state ADP concentration, which is altered by various amounts of pyruvate kinase at constant ATPase level. The half-maximal rate of stationary ATP hydrolysis is reached at an ADP concentration of 8 x 10(-6) M. The kinetic scheme is proposed explaining the requirement of the reaction products (ADP and Pi), the substrates of ATP synthesis, in the medium for proton-translocating ATP hydrolysis by P. denitrificans Fo.F1-ATP synthase.

Adenosine Diphosphate↗

PubMed2003

Sulfur oxidation in Paracoccus pantotrophus: interaction of the sulfur-binding protein SoxYZ with the dimanganese SoxB protein.

The central protein of the sulfur-oxidizing enzyme system of Paracoccus pantotrophus, SoxYZ, formed complexes with subunits associated and covalently bound. In denaturing SDS-polyacrylamide gel electrophoresis (PAGE) SoxY migrated at 12 and SoxZ at 16kDa. SDS-PAGE of homogeneous SoxYZ without reductant separated dimeric complexes of 25, 29, and 32kDa identified by the N-terminal amino acid sequences as SoxY-Y, SoxY-Z, and SoxZ-Z, and subunit cleavage by reduction suggested their linkage via protein disulfide bonds. SoxYZ was reversibly redox active between -0.25 and 0.2V, as monitored by a combined electrochemical and FTIR spectroscopic approach. The dimanganese SoxB protein (58.611Da) converted the covalently linked heterodimer SoxY-Z to SoxYZ with associated subunits which in turn aggregated to the heterotetramer Sox(YZ)(2). This reaction depended on time and the SoxB concentration, and demonstrated the interaction of these two Sox proteins.

Amino Acid Sequence↗

PubMed2005

Structural insight into SoxC and SoxD interaction and their role in electron transport process in the novel global sulfur cycle in Paracoccus pantotrophus.

Microbial oxidation of reduced inorganic sulfur compounds mainly sulfur anions in the environment is one of the major reactions of the global sulfur cycle mediated by phylogenetically diverse prokaryotes. The sulfur oxidizing gene cluster (sox) of alpha-Proteobacteria comprises of at least 16 genes, which form two transcriptional units, viz., soxSRT and soxVWXYZABCDEFGH. Sequence analysis reveals that soxD gene product (SoxD) belongs to the di-heme cytochrome c family of electron transport proteins whereas soxC gene product (SoxC) is a sulfur dehydrogenase. We employed homology modeling to construct the three-dimensional structures of the SoxC and SoxD from Paracoccus pantotrophus. SoxD protein is known to interact with SoxC. With the help of docking studies we have identified the residues involved in the interaction of SoxC and SoxD. The putative active site geometries of these two proteins as well as the structural basis of the involvements of these proteins in electron transport process during the oxidation of sulfur anions are also investigated.

Amino Acid Sequence↗

PubMed2006

Effect of electromagnetic fields on the denitrification activity of Paracoccus denitrificans.

Enzymatic activity (denitrification) of Paracoccus denitrificans was estimated electrochemically by reduction of duroquinone (DQ). Graphite electrodes covered with whole bacterial cells behind a dialysis membrane were used for measurement. P. denitrificans reduce nitrate and/or nitrite under anaerobic conditions to nitrogen gas. DQ acts as an electron mediator. After donation of the electrons to the respiratory system of the bacteria, produced DQ is reduced to durohydroquinone on the electrode surface electrocatalytically. P. denitrificans were exposed to low-frequency magnetic field (10 mT, 50 Hz) for 24 min. In comparison with the control samples, the reduction peak of I-E curves that represent denitrification activity of the cells decreased significantly after magnetic field exposure. The decrease of the peak current was about 20%. The CFU-colony forming units-method was used to estimate the number of surviving bacteria. After 24 min exposure of 10 mT magnetic field P. denitrificans culture on electrode indicates 21% bacterial death.

Colony-Forming Units Assay↗