The kinetics of electron transfer between pseudomonas aeruginosa cytochrome c-551 and its oxidase

Abstract

The redox reaction between cytochrome c-551 and its oxidase from the respiratory chain of pseudomonas aeruginosa was studied by rapid-mixing techniques at both pH7 and 9.1. The electron transfer in the direction of cytochrome c-551 reduction, starting with the oxidase in the reduced and CO-bound form, is monophasic, and the governing bimolecular rate constants are 1.3(+/- 0.2) x 10(7) M-1 . s-1 at pH 9.1 and 4 (+/- 1) x 10(6) M-1 . s-1 at pH 7.0. In the opposite direction, i.e. mixing the oxidized oxidase with the reduced cytochrome c-551 in the absence of O2, both a lower absorbance change and a more complex kinetic pattern were observed. With oxidized azurin instead of oxidized cytochrome c-551 the oxidation of the c haem in the CO-bound oxidase is also monophasic, and the second-order rate constant is 2 (+/- 0.7) x 10(6) M-1 . s-1 at pH 9.1. The redox potential of the c haem in the oxidase, as obtained from kinetic titrations of the completely oxidized enzyme with reduced azurin as the variable substrate, is 288 mV at pH 7.0 and 255 mV at pH 9.1. This is in contrast with the very high affinity observed in similar titrations performed with both oxidized azurin and oxidized cytochrome c-551 starting from the CO derivative of the reduced oxidase. It is concluded that: (i) azurin and cytochrome c-551 are not equally efficient in vitro as reducing substrates of the oxidase in the respiratory chain of Pseudomonas aeruginosa; (ii) CO ligation to the d1 haem in the oxidase induces a large decrease (at least 80 mV) in the redox potential of the c-haem moiety.

Fast Dissociation of Nitric Oxide from Ferrous Pseudomonas aeruginosa cd1 Nitrite Reductase

“…Although the biphasicity is not understood at present, it might be related to the dimeric structure of the enzyme, which has been previously shown to display cooperative behavior with some ligands, such as CO (20) and cyanide (24). An intrinsic functional asymmetry of the enzyme is also evident in the reaction with macromolecular electron donors (such as azurin and cytochrome c551) (27,28) and in the intramolecular electron transfer step (29).…”

Metalloenzymes of the denitrification pathway

“…In the presence of excess of reductant the cytochrome cd 1 nitrite reductase is inhibited by the formation of a stable complex between NO and the reduced form of heme d 1 [27,58,63]. In spite of the differences in the 3D structure of the oxidized form of both enzymes the reduced form and the one bound to the substrate and product are very similar, suggesting that one or both oxidized forms are resting states that do not participate in the catalysis [64].…”

Two c-Type Cytochromes, NirM and NirC, Encoded in the nir Gene Cluster of Pseudomonas aeruginosa Act as Electron Donors for Nitrite Reductase

“…NirM has been identified as cytochrome c 551 , the well characterized redox partner of NIR. A blue copper protein azurin has been also reported to act as an electron donor for NIR (17,19,20). nirC, but nirM, was also located in the nir gene cluster of P. denitrificans, which has cytochrome cd 1 type NIR (7).…”