A mutant of Paracoccus denitrificans deficient in cytochrome a + a 3 (original) (raw)
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Mutants of Paracoccus denitrificans Deficient in c-type Cytochromes
Journal of General Microbiology, 1979
Eight mutants of Paracoccus denitrcjicans deficient in c-type cytochrome were detected by their inability to catalyse the conversion of a-naphthol plus dimethyl-p-phenylenediamine to indophenol. From the properties of two of these mutants which completely lacked spectrophotometrically detectable c-type cytochrome, evidence was provided for the presence of a respiratory pathway which terminates in a cytochrome o-like oxidase. The presence of this pathway allows mutants lacking c-type cytochrome to grow aerobically, and also explains many of the non-mitochondria1 features of the aerobic respiratory chain of the wild-type. None of the mutants had significantly lower levels of b-type cytochromes compared with the parental strain, but two mutants were deficient in cytochrome aa3. All of the mutants could reduce nitrate, but four of them were unable to denitrify and these showed poor growth with nitrate as added terminal electron acceptor. Dundee D D l 4HN. Whiteknights, Reading RG6 2AS.
Electron transport reactions in a cytochrome c-deficient mutant of Paracoccus denitrificans
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1989
A mutant of Paracoccus denitriflcans which is deficient in c-type cytochromes grows aerobically with generation times similar to those obtained with a wild-type strain. The aa3-type oxidase is functional in the mutant as judged by spectrophotometric assays of cytochrome c oxidation using the membrane particles and cytochrome aa 3 reduction in whole cells. The cytochrome c oxidase (aa 3-tYpe) of the c-less mutant oxidizes soluble cytochrome c at rates equivalent to those obtained with the wild-type. NADH and succinate oxidase activities of the membrane preparations of the mutant and wild-type are also comparable in the absence of detergent treatment. Exogenous soluble cytochrome c can be both reduced by NADH-and succinate-linked systems and oxidized by cytochrome aa 3 present in membranes of the mutant strain. Rapid overall electron transport can occur in the c-less mutant, suggesting that reactions result from collision of diffusing complexes.
Journal of Bacteriology, 2001
Paracoccus denitrificans strains with mutations in the genes encoding the cytochrome c 550 , c 552 , or c 1 and in combinations of these genes were constructed, and their growth characteristics were determined. Each mutant was able to grow heterotrophically with succinate as the carbon and free-energy source, although their specific growth rates and maximum cell numbers fell variably behind those of the wild type. Maximum cell numbers and rates of growth were also reduced when these strains were grown with methylamine as the sole free-energy source, with the triple cytochrome c mutant failing to grow on this substrate. Under anaerobic conditions in the presence of nitrate, none of the mutant strains lacking the cytochrome bc 1 complex reduced nitrite, which is cytotoxic and accumulated in the medium. The cytochrome c 550 -deficient mutant did denitrify provided copper was present. The cytochrome c 552 mutation had no apparent effect on the denitrifying potential of the mutant cells....
Cytochrome-c-binding site on cytochrome oxidase in Paracoccus denitrificans
European journal of biochemistry / FEBS, 1998
To monitor the docking site for cytochrome c on cytochrome oxidase from Paracoccus denitrificans, a series of site-directed mutants in acidic residues exposed on the three largest subunits was constructed, and the purified enzymes were assayed for their steady-state kinetic parameters, their ionic strength dependence, and their fast electron entry kinetics by stopped-flow measurements. Increasing the ionic strength, the maximum of the bell-shaped dependence of the steady-state rate observed for wild type shifts the maximum to lower ionic strength in most of the mutants. The Km determined in steady-state experiments under different conditions is largely increased for most of the subunit II and one of the subunit I mutants, giving evidence that binding is impaired, whereas subunit III residues do not seem to contribute significantly. In addition, the bimolecular rate constant for cytochrome c oxidation under pre-steady state conditions was measured using stopped flow spectroscopy. Tak...
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1998
Cytochrome c oxidase from Paracoccus denitrificans has been purified in two different forms differing in polypeptide composition. An enzyme containing polypeptides I^IV is obtained when the purification procedure is performed in L-Ddodecylmaltoside. If, however, Triton X-100 is used to purify the enzyme under otherwise identical conditions, an enzyme is obtained containing only subunits I^II. The two enzymes are undistinguishable by optical spectroscopy but show significant differences in the transient and steady-state time regimes, as studied by stopped-flow spectroscopy. The observed differences, however, are not due to removal of subunits III and IV, but rather to a specific effect of Triton X-100 which appears to affect cytochrome c binding. From these results it is not expected that subunits III and IV play any significant role in cytochrome c binding and, possibly, in the subsequent electron transfer processes. The results also suggest that both electrostatic and hydrophobic interactions may be important in the initial electron transfer process from cytochrome c.
Biochemistry, 2000
Heme-copper terminal oxidases use the free energy of oxygen reduction to establish a transmembrane proton gradient. While the molecular mechanism of coupling electron transfer to proton pumping is still under debate, recent structure determinations and mutagenesis studies have provided evidence for two pathways for protons within subunit I of this class of enzymes. Here, we probe the D-pathway by mutagenesis of the cytochrome c oxidase of the bacterium Paracoccus denitrificans; amino acid replacements were selected with the rationale of interfering with the hydrophilic lining of the pathway, in particular its assumed chain of water molecules. Proton pumping was assayed in the reconstituted vesicle system by a stopped-flow spectroscopic approach, allowing a reliable assessment of proton translocation efficiency even at low turnover rates. Several mutations at positions above the cytoplasmic pathway entrance (Asn 131, Asn 199) and at the periplasmic exit region (Asp 399) led to complete inhibition of proton pumping; one of these mutants, N131D, exhibited an ideal decoupled phenotype, with a turnover comparable to that of the wild-type enzyme. Since sets of mutations in other positions along the presumed course of the pathway showed normal proton translocation stoichiometries, we conclude that the D-pathway is too wide in most areas above positions 131/199 to be disturbed by single amino acid replacements.
Cytochrome c′ of Paracoccus denitrificans
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1991
Cytochrome c' was identified in periplasmic extracts of the Paracoccus denitrificans strains LMD 22.21 and LMD 52.44. The cytochrome c' was purified from the latter using the device of sequential molecular exclusion chromatography in the dimeric and monomeric states. Although showing the overall spectroscopic features of the cytochrome c' family, the Paracoccus cytochrome c' is unusual in having a red-shifted oxidised Soret band at 407 nm. Also unusual is the midpoint potential of 202 mV, well above the known cytochrome c' range. The amino-acid composition of Pa. denitrificans cytochrome c' showed the high alanine and low proline content characteristic of the group and reflecting the predominantly a-helical character of the protein. Comparison of the amino-acid compositions suggests some similarity to the cytochromes c' of Chromatium vinosum and halotolerant Paracoccus.