Cytochrome c-551 and azurin oxidation catalysed by Pseudomonas aeruginosa cytochrome oxidase. A steady-state kinetic study

Abstract

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.

Cytochrome cd1, Reductive Activation and Kinetic Analysis of a Multifunctional Respiratory Enzyme

“…2) compared with ϳ8 s Ϫ1 (29) for the P. aeruginosa protein at the same pH. Similarly, we observed a K M for oxygen of 160 M for P. pantotrophus cd 1 , in marked contrast to the values as low as 2 M reported for P. aeruginosa cd 1 (29,36). Some of these variances might arise from differences in experimental conditions, but the trend seems to suggest true differences in the functionality of the enzymes, such that there may be two distinct classes of cytochrome cd 1 -type nitrite reductase.…”

Coupling of Protonation, Reduction, and Conformational Change in azurin from Pseudomonas aeruginosa Investigated with Free Energy Measures of Cooperativity

“…PaAz was believed to function as electron donor to cytochrome cd 1 nitrite reductase. 1,88,89 The gene for azurin was, however, later found not to be essential for denitrification 3 and not located close to other denitrification genes in the genome of P. aeruginosa. 90 Instead, two soluble cytochromes c from the nitrite reductase gene cluster were found also to be able to donate electrons to nitrite reductase at similar rates.…”

The nitrite reductase from Pseudomonas aeruginosa : Essential role of two active-site histidines in the catalytic and structural properties

“…Cytochrome oxidase activity was assessed at 20°C in 50 mM sodium phosphate buffer (pH 7.0) by measuring the rate of oxidation of reduced P. aeruginosa cytochrome c 551 (1-20 M) as described in ref. 11. NIR activity was measured anaerobically at 27°C in 50 mM sodium phosphate buffer (pH 6.2), either after oxidation of reduced azurin (12) or during amperometric measurement of NO production with a NO electrode (ISO-NO; World Precision Instruments, Sarasota, FL).…”