Some spectral and steady-state kinetic properties of Pseudomonas cytochrome oxidase

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Abstract
Some spectra of Pseudomonas cytochrome oxidase [EC 1.9.3.2] are reported, both for comparison with those of other workers and to illustrate the differences between the ascorbate- and dithionite-reduced forms of the enzyme. A spectrum of the reduced enzyme-CO complex, prepared in the absence of added reductants by incubation under CO, is also included. Ultracentrifugation studies yielded a value for the sedimentation coefficient (s20,w) of 7.5 S and an isoelectric point of pH 6.9 was determined by isoelectric focusing. Steady-state kinetic constants of the electron donors, quinol, sodium ascorbate, reduced Pseudomonas azurin and Pseudomonas ferrocytochrome c551 were investigated giving Km values of 30 mM, 4 mM, 49 .mu.M and 5.6 .mu.M, respectively. The 2 protein substrates were observed to be subject to product inhibition and the Ki for oxidized Pseudomonas azurin was evaluated at 4.9 .mu.M. Steady-state kinetics were also used to investigate the effects of the oxidation products of dithionite on the oxidase and nitrite reductase activities of Pseudomonas cytochrome oxidase. Whereas the oxidase activity was inhibited, the nitrite reductase activity was slightly enhanced.