A mutant of Paracoccus denitrificans deficient in cytochrome a + a 3 (original) (raw)
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.
Molecular Microbiology, 1996
SummaryIn Paracoccusdenitrificans the aa3‐type cytochrome c oxidase and the bb3‐type quinol oxidase have previously been characterized in detail, both biochemically and genetically. Here we report on the isolation of a genomic locus that harbours the gene cluster ccoNOQP, and demonstrate that it encodes an alternative cbb3‐type cytochrome c oxidase. This oxidase has previously been shown to be specifically induced at low oxygen tensions, suggesting that its expression is controlled by an oxygen‐sensing mechanism. This view is corroborated by the observation that the ccoNOQP gene cluster is preceded by a gene that encodes an FNR homologue and that its promoter region contains an FNR‐binding motif. Biochemical and physiological analyses of a set of oxidase mutants revealed that, at least under the conditions tested, cytochromes aa3, bb3. and cbb3 make up the complete set of terminal oxidases in P. denitrificans. Proton‐translocation measurements of these oxidase mutants indicate that ...
The role of tryptophan 272 in theParacoccus denitrificanscytochromecoxidase
FEBS Letters, 2006
The mechanism of electron coupled proton transfer in cytochrome c oxidase (CcO) is still poorly understood. The P Mintermediate of the catalytic cycle is an oxoferryl state whose generation requires one additional electron, which cannot be provided by the two metal centres. The missing electron has been suggested to be donated to this binuclear site by a tyrosine residue. A tyrosine radical species has been detected in the P M and F Å intermediates (formed by addition of H 2 O 2) of the Paraccocus denitrificans CcO using electron paramagnetic resonance (EPR) spectroscopy. From the study of conserved variants its origin was determined to be Y167 which is surprising as this residue is not part of the active site. Upon inspection of the active site it becomes evident that W272 could be the actual donor of the missing electron, which can then be replenished from Y167 or from the Y280-H276 cross link in the natural cycle. To address the question, whether such a direct electron transfer pathway to the binuclear centre exists two tryptophan 272 variants in subunit I have been generated. These variants are characterised by their turnover rates as well as using EPR and optical spectroscopy. From these experiments it is concluded, that W272 is an important intermediate in the formation of the radical species appearing in P M and F Å intermediates produced with hydrogen peroxide. The significance of this finding for the catalytic function of the enzyme is discussed.
The kinetics and stoichiometry of the redox-linked protonation of the soluble Paracoccus denitrificans cytochrome c oxidase were investigated at pH ) 7.2-7.5 by multiwavelength stopped-flow spectroscopy, using the pH indicator phenol red. We compared the wild-type enzyme with the K354M and the D124N subunit I mutants, in which the K-and D-proton-conducting pathways are impaired, respectively. Upon anaerobic reduction by Ru-II hexamine, the wild-type enzyme binds 3.3 ( 0.6 H + / aa 3 , i.e., approximately 1 H + in excess over beef heart oxidase under similar conditions and the D124N mutant 3.2 ( 0.5 H + /aa 3 . In contrast, in the K354M mutant, in which the reduction of heme a 3 -Cu B is severely impaired, ∼0.8 H + is promptly bound synchronously with the reduction of heme a, followed by a much slower protonation associated with the retarded reduction of the heme a 3 -Cu B site. These results indicate that complete reduction of heme a (and Cu A ) is coupled to the uptake of ∼0.8 H + , which is independent of both H + -pathways, whereas the subsequent reduction of the heme a 3 -Cu B site is associated with the uptake of ∼2.5 H + transferred (at least partially) through the K-pathway. On the basis of these results, the possible involvement of the D-pathway in the redox-linked protonation of cytochrome c oxidase is discussed. † Work partially supported by Ministero dell'Istruzione, dell'Università e della Ricerca of Italy (PRIN "Bioenergetica: genomica funzionale, meccanismi molecolari ed aspetti fisiopatologici" and Fondo per gli Investimenti della Ricerca di Base RBAU01F2BJ to P.S., Progetto Giovani Ricercatori 2002 to A.G.), and by Deutsche Forschungsgemeinschaft (SFB 472 to B.L.).
Biochimica et Biophysica Acta (BBA) - Bioenergetics, 2011
Cytochrome bc 1 complex Complex III Cytochrome bc 1 Δac complex Laser flash photolysis Steady state kinetic The cytochrome bc 1 complex is a key component in several respiratory pathways. One of the characteristics of the eukaryotic complex is the presence of a small acidic subunit, which is thought to guide the interaction of the complex with its electron acceptor and facilitate electron transfer. Paracoccus denitrificans represents the only example of a prokaryotic organism in which a highly acidic domain is covalently fused to the cytochrome c 1 subunit. In this work, a deletion variant lacking this acidic domain has been produced and purified by affinity chromatography. The complex is fully intact as shown by its X-ray structure, and is a dimer (Kleinschroth et al., subm.) compared to the tetrameric (dimer-of-dimer) state of the wild-type. The variant complex is studied by steady-state kinetics and flash photolysis, showing wild type turnover and a virtually identical interaction with its substrate cytochrome c 552 .
Deletion of the gene for subunit III leads to defective assembly of bacterial cytochrome oxidase
The EMBO Journal, 1989
Communicated by M.Wikstrom COIH is one of the major subunits in the mitochondrial and a bacterial cytochrome c oxidase, cytochrome aa3. It does not contain any of the enzyme's redox-active metal centres and can be removed from the enzyme without major changes in its established functions. We have deleted the COmI gene from Paracoccus denitificans. The mutant still expresses spectroscopically detectable enzyme almost as the wild-type, but its cytochrome c oxidase activity is much lower. From 50 to 80% of cytochrome a is reduced and its absorption maximum is 2-3 nm blueshifted. The EPR signal of ferric cytochrome a is heterogeneous indicating the presence of multiple cytochrome a species. Proteolysis of the membrane-bound oxidase shows new cleavage sites both in COI and COII. DEAE-chromatography of solubilized enzyme yields fractions that contain a COI + COIH complex and in addition haem-binding, free COI as well as free COII. The mutant phenotype can be complemented by introducing the COIH gene back to cells in a plasmid vector. We conclude that cytochrome oxidase assembles inefficiently in the absence of COm and that this subunit may facilitate a late step in the assembly. The different oxidase species in the mutant represent either accumulating intennediates of the assembly pathway or dissociation products of a labile COI + COIl complex and its conformational variants.
Genes coding for cytochromec oxidase inParacoccus denitrificans
Journal of Bioenergetics and Biomembranes, 1991
Several loci on the Paracoccus denitrificans chromosome are involved in the synthesis of cytochrome c oxidase. So far three genetic loci have been isolated. One of them contains the structural genes of subunits II and III, as well as two regulatory genes which probably code for oxidase-specific assembly factors. In addition, two distinct genes for subunit I have been cloned, one of which is located adjacent to the cytochrome c»50 gene. An alignment of six promoter regions reveals only short common sequences.
The role of tryptophan 272 in the Paracoccus denitrificans cytochrome c oxidase
FEBS Letters, 2006
The mechanism of electron coupled proton transfer in cytochrome c oxidase (CcO) is still poorly understood. The PM‐intermediate of the catalytic cycle is an oxoferryl state whose generation requires one additional electron, which cannot be provided by the two metal centres. The missing electron has been suggested to be donated to this binuclear site by a tyrosine residue. A tyrosine radical species has been detected in the PM and F intermediates (formed by addition of H2O2) of the Paraccocus denitrificans CcO using electron paramagnetic resonance (EPR) spectroscopy. From the study of conserved variants its origin was determined to be Y167 which is surprising as this residue is not part of the active site. Upon inspection of the active site it becomes evident that W272 could be the actual donor of the missing electron, which can then be replenished from Y167 or from the Y280‐H276 cross link in the natural cycle. To address the question, whether such a direct electron transfer pathway...