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PubMed2005

Multifrequency EPR analysis of the dimanganese cluster of the putative sulfate thiohydrolase SoxB of Paracoccus pantotrophus.

A detailed analysis of the EPR signatures at X-band and Q-band of an enzyme (SoxB) involved in sulfur oxidation from Paracoccus pantotrophus is presented. EPR spectra are attributed to an exchange-coupled dimanganese Mn(2)(II,II) complex. An antiferromagnetic exchange interaction of J=-7.0 (+/-1) cm(-1) (H=-2JS ( 1 ) S ( 2 )) is evidenced by a careful examination of the temperature dependence of the EPR spectra. The spin Hamiltonian parameters for a total spin of S ( T ) =1, 2 and 3 are obtained and an inter-manganese distance of 3.4 (+/-0.1) A is estimated. The comparison with exchange coupling and inter-manganese distance data of other dimanganese proteins and model compounds leads to a tentative assignment of the Mn bridging ligands to bis(mu-hydroxo) (mu-carboxylato).

Electron Spin Resonance Spectroscopy↗

PubMed2000

ESEEM studies of succinate:ubiquinone reductase from Paracoccus denitrificans.

Electron spin-echo envelope modulation (ESEEM) spectroscopy has been performed in order to obtain structural information about the environment of the reduced [2Fe-2S] cluster (S-1 center), the oxidized [3Fe-4S] cluster (S-3 center), and the flavin semiquinone radical in purified succinate:ubiquinone reductase from Paracoccus denitrificans. Spectral simulations of the ESEEM data from the reduced [2Fe-2S] yielded nuclear quadrupole interaction parameters that are indicative of peptide nitrogens. We also observed a weak interaction between the oxidized [3Fe-4S] cluster and a peptide 14N. There was no evidence for coordination of any of the Fe atoms to 14N atoms of imidazole rings. The ESEEM data from the flavin semiquinone radical were more complicated. Here, evidence was obtained for interactions between the unpaired electron and only the two nitrogen atoms in the flavin ring.

Animals↗

PubMed2005

Introducing a novel human mtDNA mutation into the Paracoccus denitrificans COX I gene explains functional deficits in a patient.

We identified a novel mutation (S142F) in the human mtDNA CO I gene in a patient with a clinical phenotype resembling mitochondrial cardioencephalomyopathy. To substantiate pathogenicity, we modeled the identified mutation in the homologous gene in Paracoccus denitrificans and analyzed the biochemical consequences. We observed a deleterious effect on enzyme activity, with a lack of heme a3. Taking advantage of the extensive structural homology between the bacterial enzyme and the mammalian core complex, we conclude that the novel S142F mutation is disease-related. This approach can be used in other cases to support the pathogenicity of novel variants in the mitochondrial genome.

Adult↗

PubMed2006

Functional expression of the astaxanthin biosynthesis genes from a marine bacterium, Paracoccus haeundaensis.

The astaxanthin biosynthesis gene cluster in Paracoccus haeundaensis consists of six genes: crtW, crtZ, crtY, crtI, crtB, and crtE contain 726, 486, 1158, 1503, 912, and 879 base pairs, respectively. Individual carotenoid biosynthesis genes of P. haeundaensis have now been expressed in E. coli and each gene product has been purified to homogeneity. Their molecular characteristics, including enzymatic activities, are reported here.

Carotenoids↗

PubMed1987

Is the ubiquinone pool in the respiratory chain of the bacterium Paracoccus denitrificans really unhomogeneous?

We have established the participation of a mobile redox pool in the respiratory chain of anaerobically grown bacterium Paracoccus denitrificans. In testing the kinetical homogeneity of the pool it was found that the ratio of fluxes of electron transport toward the terminal acceptors oxygen and nitrate was coincident for the respiratory substrates NADH and succinate; this provides evidence against the preferential link of one dehydrogenase with a distinct terminal enzyme through the separate pool of ubiquinone. The deviation from the expected behavior observed in comparing the titration of NADH oxidase and succinate oxidase with respiratory inhibitors such as mucidin (inhibitor in the bc1 region) or cyanide can be accounted for by the activation of succinate dehydrogenase upon the increase in the reduced state of respiratory components during the titration.

Alkenes↗

PubMed1977

Tightly bound nucleotides of the energy-transducing ATPase, and their role in oxidative phosphorylation. I. The Paracoccus denitrificans system.

1. The coupling ATPase of Paracoccus denitrificans can be removed from the membrane by washing coupled membrane fragments at low salt concentrations. 2. This ATPase resembles coupling ATPases of mitochondria, chloroplasts and other bacteria. It is a negatively charged protein of molecular weight about 300,000. An inhibitor protein in bound tightly to the ATPase in vivo, and can be destroyed by trypsin treatment. 3. ATP and ADP are found tightly bound to the coupling ATPase of P. denitrificans, both in its membrane-bound and isolated state. The ATP/ADP ratio on the enzyme is greater than one. 4. Under de-energised condtions, the bound nucleotides are not available to the suspending medium. When the membrane is energised however, the bound nucleotides can exchange with added nucleotides and incorporate 32Pi. 32Ppi is incorporated into the beta and gamma positions of the bound nucleotides, but beta-labelling probably does not occur on the coupling ATPase. 5. Uncouplers inhibit the exchange of the free nucleotides or 32Pi into the bound nucleotides, while venturicidin (an energy transfer inhibitor) and aurovertin stimulate the exchange. 6. The response of the bound nucleotides to energisation is consistent with their being involved directly in the mechanism of oxidative phosphorylation.

Adenosine Diphosphate↗

PubMed1980

A comparison of the respiratory chain in particles from Paracoccus denitrificans and bovine heart mitochondria by EPR spectroscopy.

A study is presented on the EPR characteristics of the paramagnetic groups in the respiratory chain present in membrane particles of Paracoccus denitrificans, the respiratory system of which is very similar to that in submitochondrial particles from beef heart. All paramagnetic prosthetic groups of the mitochondrial system are also found in the bacterial plasma membrane. Their properties suggest that the respiratory groups are embedded in very similar protein environments in the two systems.

Animals↗

PubMed1980

A specific uncoupler-binding protein in Tetrahymena pyriformis and Paracoccus denitrificans.

The uncoupler of mitochondrial oxidative phosphorylation, 2-nitro-4-azido-carbonylcyanide phenylhydrazone (N3CCP) which is capable of photoaffinity labeling has been used to examine the effect of uncouplers on the energy conserving membrane of Paracoccus denitrificans and Tetrahymena pyriformis. The N3CCP uncouples respiration in P. denitrificans and T. pyriformis cells with U1/2 values of 1.05 microM and 0.24 microM, respectively. Binding studies show the presence of 0.65 +/- 0.05 high affinity sites per cytochrome alpha with Kd of 0.5 +/- 0.1 microM in P. denitrificans membranes and 1.4 +/- 0.2 sites per cytochrome alpha 2 with a Kd of 0.4 +/- 0.1 microM in T. pyriformis membranes. Irradiation of [3H]-N3CCP bound to the membranes leads to a covalent linking of the radioactive uncoupler to a peptide of 10--15 kdaltons as analyzed by SDS-polyacrylamide gel electrophoresis. It is concluded that these two microbial systems contain a specific high affinity uncoupler binding site very similar to that of mammalian mitochondria (Katre, N.V. and Wilson, D.F. (1978) Arch. Biochem. Biophys. 191, 647--656).

Acetonitriles↗

PubMed1981

Copper and manganese electron spin resonance studies of cytochrome c oxidase from Paracoccus denitrificans.

The two-subunit cytochrome c oxidase from Paracoccus denitrificans contains two heme a groups and two copper atoms. However, when the enzyme is isolated from cells grown on a commonly employed medium, its electron paramagnetic resonance (EPR) spectrum reveals not only a Cu(II) powder pattern, but also a hyperfine pattern from tightly bound Mn(II). The pure Mn(II) spectrum is observed at -40 degrees C; the pure Cu(II) spectrum can be seen with cytochrome c oxidase from P. denitrificans cells that had been grown in a Mn(II)-depleted medium. This Cu(II) spectrum is very similar to that of cytochrome c oxidase from yeast or bovine heart. Manganese is apparently not an essential component of P. denitrificans cytochrome c oxidase since it is present in substoichometric amounts relative to copper or heme a and since the manganese-free enzyme retains essentially full activity in oxidizing ferrocytochrome c. However, the manganese is not removed by EDTA and its EPR spectrum responds to the oxidation state of the oxidase. In contrast, manganese added to the yeast oxidase or to the manganese-free P. denitrificans enzyme can be removed by EDTA and does not respond to the oxidation state of the enzyme. This suggests that the manganese normally associated with P. denitrificans cytochrome c oxidase is incorporated into one or more internal sites during the biogenesis of the enzyme.

Animals↗

PubMed1981

Proton pump coupled to cytochrome c oxidase in Paracoccus denitrificans.

The proton translocating properties of cytochrome c oxidase in whole cells of Paracoccus denitrificans have been studied with the oxidant pulse method. leads to H+/2e- quotients have been measured with endogenous substrates, added methanol and added ascorbate (+TMPD) as reductants, and oxygen and ferricyanide as oxidants. It was found that both the observed leads to H+/O with ascorbate (+TMPD) as reductant, and the differences in proton ejection between oxygen-and ferricyanide pulses, with endogenous substrates or added methanol as a substrate, indicate that the P. denitrificans cytochrome c oxidase translocates protons with a stoichiometry of 2H+/2e-. The results presented in this and previous papers are in good agreement with recent findings concerning the mitochondrial cytochrome c oxidase, and suggest unequal charge separation by different coupling segments of the respiratory chain of P. denitrificans.

Anaerobiosis↗

PubMed1981

Midpoint potentials of cytochromes in vesicles of anaerobically-grown Paracoccus denitrificans determined by the indirect coulometric titration method.

1. Multiplicity of redox components with spectral properties similar to b-type cytochromes was established in vesicles derived fro anaerobically-grown Paracoccus denitrificans. 2. Multiplicity of c-type cytochromes was not apparent either from low temperature spectroscopy or potentiometric titrations. 3. Cytochromes a + a3 and a component, only observable at liquid nitrogen temperature, with a spectral maximum at 582.5 nm were detected. 4. Redox cycling of electron transport components using the indirect coulometric titration method was a convenient means of pairing redox potentials and was reproducible in total absorbance changes, midpoint potentials and spectral maxima.

Anaerobiosis↗

PubMed1981

Respiration-driven proton translocation with nitrite and nitrous oxide in Paracoccus denitrificans.

(1)H+ leads to/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O2, NO2- or N2O. Under optimal H+-translocation conditions, the ratios leads to H+/O, H+ leads to/N2O, H+ leads to/NO2- for reduction to N2 and H+ leads to/NO2- for reduction to N2O were 6.0-6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N,'N'-tetramethyl-p-phenylene-diamine as exogenous substrate, addition of NO2- or N2O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. H+/N2O, H+/NO2- for reduction to N2 and H+/NO2- for reduction to N2O were -0.84, -2.33 and -1.90, respectively. (3) The H+/oxidant ratios, mentioned in item 2, were not altered in the presence of valinomycin/K+ and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O2, NO2- and N2O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO2-, N2O or O2 pass two H+-trans-locating sites. The H+ leads to/electron acceptor ratios predicted by this scheme are in good agreement with the experimental values.

Electron Transport↗

PubMed1983

Reaction of cytochrome c in the electron-transport chain of Paracoccus denitrificans.

The reaction of the cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) of Paracoccus denitrificans cytoplasmic membranes with the endogenous cytochrome c of the membranes was studied, as well as its interaction with added exogenous cytochrome c from P. denitrificans or bovine heart. The polarographic method was employed, using N,N,N',N'-tetramethyl-p-phenylenediamine plus ascorbate to reduce the cytochrome c. We found that overall electron transport can proceed maximally while the cytochrome c remains membrane bound; NADH or succinoxidase activities were not inhibited by the addition of substances which bind the P. denitrificans cytochrome c strongly. In contrast to our observations with the spectrophotometric method (Smith, L., Davies, H.C. and Nava, M.E. (1976) Biochemistry 15, 5827-5831), in the polarographic assays the membrane-bound oxidase reacts with about equal rapidity with exogenous bovine and P. denitrificans cytochromes c. The reaction of the oxidase with the endogenous cytochrome c proceeds at high rates and preferentially to that with exogenous cytochrome c; the reaction with the latter, but not the former is inhibited by positively charged poly(L-lysine). The cytochrome c and the oxidase appear to be very closely associated on the membrane.

Animals↗

PubMed1984

Effects of monoclonal antibodies to bovine and Paracoccus denitrificans cytochromes c on reactions with oxidase, reductase and peroxidase.

The effects of monoclonal antibodies to bovine and Paracoccus denitrificans cytochromes c (Kuo, L.M. and Davies, H.C. (1983) Mol. Immunol. 20, 827-838) in the reactions of the cytochromes c with cytochrome c oxidase, reductase and peroxidase were studied. Spectrophotometric assays were employed, under conditions where binding of cytochrome c to the enzymes appears to be rate-limiting. Less than stoichiometric amounts of antibodies to P. denitrificans cytochrome c added to the cytochrome rendered some of it nonoxidizable or nonreducible by the P. denitrificans membrane-bound electron transport system and decreased the rate constant with the remaining cytochrome c. The antibodies appear to affect both electron transport reactions (blocking effects) with the oxidase and reductase and binding effects (effects on rate constants) and to distinguish between the two. Different ratios of antibody site to cytochrome c gave different extents of blocking of the reductase as compared with the oxidase reaction. Differences were also apparent in the effect of these antibodies on the reaction of yeast peroxidase and the oxidase with the P. denitrificans cytochrome c. Antibodies to bovine and P. denitrificans cytochromes c had considerably less effect on the reactions of the bovine cytochrome with bovine oxidase and reductase. One antibody was inhibitory to the oxidase reaction with bovine cytochrome c, but not to that with the reductase. Also, an antibody which inhibited the oxidase reaction had no effect on the reaction with yeast peroxidase. The data give evidence that the interaction areas on cytochrome c for oxidase and reductase and peroxidase are not identical, although they may be nearby.

Animals↗

PubMed1985

Monoclonal antibodies to cytochrome c from Paracoccus denitrificans: effects on electron transport reactions.

The effect of a monoclonal antibody to a soluble cytochrome c from Paracoccus denitrificans was tested on the membrane-bound electron-transport system of this bacterium. This antibody (F3-10.2) and one previously described (F3-29.4) (Kuo, L.M., Davies, H.C. and Smith, L. (1984) Biochim. Biophys. Acta 766, 472-482) were deduced to bind to the cytochrome c in the area including amino acid residue number 23 on a loop on the side of the heme crevice. In contrast to the observations with the previously tested antibody, the present data show the second antibody to block completely the reaction of the cytochrome c with cytochrome c oxidase but not that with cytochrome c reductase. Neither antibody has an appreciable inhibitory effect on the NADH oxidase of the isolated detergent-treated membranes. The two antibodies bind in different ways, giving insight into the interaction of a soluble protein with membrane-bound enzymes. The data indicate that the reaction sites on the cytochrome c for the oxidase and reductase moieties of P. denitrificans are different. They also argue against the need for a dissociable cytochrome c comparable to that which functions on the mitochondrial inner membrane.

Antibodies, Monoclonal↗

PubMed1990

Inhibition by trimethylamine of methylamine oxidation by Paracoccus denitrificans and bacterium W3A1.

Trimethylamine, a common substrate for methylotrophic growth, specifically inhibited methylamine-dependent respiration by Paracoccus denitrificans and bacterium W3A1. These effects were caused by the specific inhibition by trimethylamine of the periplasmic quinoprotein methylamine dehydrogenase. Steady-state kinetic analysis of the effect of trimethylamine on methylamine oxidation by methylamine dehydrogenase indicated that the inhibition was a mixed type. Apparent Ki values for trimethylamine of 1.1 mM and 4.7 mM, respectively, were obtained for the P. denitrificans and bacterium W3A1 enzymes. Methylamine-dependent oxygen consumption by each bacterium was inhibited either by preincubation of cells with trimethylamine prior to the addition of substrate or by addition of trimethylamine to actively respiring cells. Formate-dependent respiration was not inhibited by trimethylamine. A scheme is proposed which describes a regulatory role for trimethylamine in the metabolism and dissimilation of methylamine by methylotrophic bacteria.

Bacteria↗

PubMed1990

Nitric oxide formed by nitrite reductase of Paracoccus denitrificans is sufficiently stable to inhibit cytochrome oxidase activity and is reduced by its reductase under aerobic conditions.

Nitric oxide, generated by the action of purified nitrite reductase, inhibited the oxidase activity of both membrane vesicles from anaerobically grown Paracoccus denitrificans and bovine heart submitochondrial particles. In the former case, the inhibition was relatively short-lived and its duration was reduced either by decreasing the concentration of nitrite or raising the ratio of vesicles to nitrite reductase enzyme. These observations indicate that nitric oxide, at least at low concentrations, was sufficiently stable in the presence of oxygen to allow diffusion between proteins in aqueous solution. The shorter inhibition period with P. denitrificans membrane vesicles implies that the nitric oxide reductase of the vesicles is active in the presence of oxygen and has a sufficiently high affinity for nitric oxide to remove it from oxidase enzymes by competition. These observations are related to previous reports of potent inhibition under certain conditions of oxidase activity of P. denitrificans cells by a molecular species produced from nitrite. The implications of the deduced stability of nitric oxide in aerobic solutions are considered with respect to both the phenomenon of aerobic denitrification and the synthesis of nitric oxide in mammalian cells.

Aerobiosis↗

PubMed1994

Influence of the lipid matrix on incorporation and function of LPS-free porin from Paracoccus denitrificans.

We have studied the role of lipopolysaccharide (LPS) for the insertion of LPS-free porin from Paracoccus denitrificans into planar lipid bilayers and its function therein. For this, we reconstituted the porin into different asymmetric planar lipid bilayers with or without LPS and into symmetric phospholipid bilayers. LPS-free porin added to the various bilayer systems was found to induce a step-wise increase in membrane conductance with different incorporation rates, depending on the presence of LPS in the bilayer leaflet opposite to porin addition. The incorporation rate into asymmetric LPS/phospholipid membranes from the phospholipid side was more than 10-fold higher than that observed for pure phospholipid membranes. The porin formed general diffusion pores without any salt specificity. The mean single-channel conductance did not depend on the presence of LPS and was about 4.2 nS for a subphase containing 1 M KCl in all systems tested. At certain applied transmembrane voltages, which depended on membrane composition and were approximately greater than 100 mV for the LPS/phospholipid system, single-channel closing in three steps was observed. Differences in the voltage dependence of porin-channel closing could be correlated with the surface charge of the bilayer. From the voltage-dependent gating behaviour proof for an oriented incorporation of the porin molecules, depending on the side of porin addition, and evidence for their orientation could be derived. Measurements at temperatures above and below the beta<==>alpha phase transition temperature of LPS gave evidence for the influence of membrane rigidity on the gating behaviour.

Carbohydrate Sequence↗