Formation and decay of the primary oxygen compound of cytochrome oxidase at room temperature as observed by stopped flow, laser flash photolysis and rapid scanning.

Rapid kinetic and scanning techniques were employed to observe the reaction of dioxygen with fully reduced cytochrome oxidase at room temperature following stopped flow flash photolysis of the CO compound of the enzyme. The initial product of the reaction was an oxygen compound with a spectral profile quite similar to that of the cytochrome oxidase-CO compound. The second order rate constant for formation of the primary oxygen compound was 1.1 X 10(8) M-1 S-1 at pH 7.4 and 25 degrees C. With an off rate constant of 1.9 X 10(3) S-1, the calculated dissociation constant was 1.8 X 10(-5) M. The apparent activation energy was 16.7 kJ/mol. The primary oxygen compound underwent further reaction which was independent of oxygen concentration with a rate constant of 1.2 X 10(3) S-1, and the product was Intermediate III ( Orii , Y. (1982) in Oxygenases and Oxygen Metabolism ( Nozaki , M., Yamamoto, S., Ishimura , Y., Coon , M. J., Ernster , L., and Estabrook , R., eds) pp. 137-149, Academic Press, New York), an oxidized species presumably identical to the "oxygen pulsed" oxidase. Thus, this reaction was ascribed to intramolecular electron transfer to the oxygen in the compound. The energy of activation was 49.8 kJ/mol below 18 degrees C but above that the rate constant was independent of temperature, and a "tunneling" mechanism was suggested for the intramolecular electron transfer.