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Cytochrome c550 from Pseudomonas aeruginosa.

ABSTRACT: In cells of Pseudomonas aeruginosa A.T.C.C. 17933 grown on ethanol the synthesis of a soluble c-type cytochrome, together with quinoprotein ethanol dehydrogenase, is induced. The cytochrome, with an alpha-absorption band at 550 nm, was purified to homogeneity. The molecular mass of the monomeric protein is 15 kDa, the pI is 4.8, and it contains one haem prosthetic group. The midpoint potential of the autoxidizable, but not autoreducible, cytochrome is 280 mV. Cytochrome c550 mediates electron transfer between quinoprotein ethanol dehydrogenase and ferricyanide. In a system composed of membrane particles with NN'NN'-tetramethyl-p-phenylenediamine oxidase activity and quinoprotein ethanol dehydrogenase, oxygen consumption is only observed in the presence of cytochrome c550. This indicates the participation of the cytochrome in the electron-transport chain linked to quinoprotein ethanol dehydrogenase in P. aeruginosa. The electron transport from ethanol dehydrogenase to oxygen is inhibited by myxothiazol and antimycin, indicating that a cytochrome bc1-like complex is involved.

SUBMITTER: Reichmann P

PROVIDER: S-EPMC1132146 | biostudies-other | 1993 Jan

REPOSITORIES: biostudies-other

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Project description:The kinetics of oxidation of azurin and cytochrome c-551 catalysed by Pseudomonas aeruginosa cytochrome oxidase were re-investigated, and the steady-state parameters were evaluated by parametric and non-parametric methods. At low concentrations of substrates (e.g. less than or equal to 50 microM) the values obtained for Km and catalytic-centre activity are respectively 15 +/- 3 microM and 77 +/- 6 min-1 for azurin and 2.15 +/- 0.23 microM and 66 +/- 2 min-1 for cytochrome c-551, in general accord with previous reports assigning to cytochrome c-551 the higher affinity for the enzyme and to azurin a slightly higher catalytic rate. However, when the cytochrome c-551 concentration was extended well beyond the value of Km, the initial velocity increased, and eventually almost doubled at a substrate concentration greater than or equal to 100 microM. This result suggests a 'half-hearted' behaviour, since at relatively low cytochrome c-551 concentrations only one of the two identical binding sites of the dimeric enzyme seems to be catalytically active, possibly because of unfavourable interactions influencing the stability of the Michaelis-Menten complex at the second site. When reduced azurin and cytochrome c-551 are simultaneously exposed to Ps. aeruginosa cytochrome oxidase, the observed steady-state oxidation kinetics are complex, as expected in view of the rapid electron transfer between cytochrome c-551 and azurin in the free state. In spite of this complexity, it seems likely that a mechanism involving a simple competition between the two substrates for the same active site on the enzyme is operative. Addition of a chemically modified and redox inactive form of azurin (Hg-azurin) had no effect on the initial rate of oxidation of either azurin and cytochrome c-551, but clearly altered the time course of the overall process by removing, at least partially, the product inhibition. The results lead to the following conclusions: (i) reduced azurin and cytochrome c-551 bind at the same site on the enzyme, and thus compete; (ii) Hg-azurin binds at a regulatory site, competing with the product rather than the substrate; (iii) the two binding sites on the dimeric enzyme, though intrinsically equivalent, display unfavourable interactions. Since water is the product of the reduction of oxygen, point (iii) has important implications for the reaction mechanism.

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Cytochrome c550 from Pseudomonas aeruginosa.

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