Abstract
The reduction of bovine heart cytochrome oxidase by the 1-methylnicotinamide (MNA) radical was investigated by the use of pulse radiolysis. With the decay of the MNA radical, the absorption at 445 and 605 nm, a characteristic to ferrous heme a of the oxidase, increased. The kinetic difference spectrum obtained was similar to that of the fully reduced minus the fully oxidized form of the oxidase, and was not different from that obtained in the reaction of the MNA radical with the mixed valence CO complex of the oxidase, where heme a3 is the CO-bound reduced form with heme a oxidized. This suggests that the absorption changes at 445 and 605 nm arise from the reduction of heme a, not heme a3. In order to elucidate the contribution of "visible" copper in this reaction, the absorption of the oxidase in the near-infrared region was measured. A decrease of the 830 nm band due to the reduction of visible copper was detected with a half-life of 5 microseconds. This absorption change obeyed pseudo-first order kinetics and its rate constant increased with the concentration of the oxidase. This suggests that the absorption change at 830 nm is followed by a bimolecular reaction of the MNA radical with visible copper of the oxidase. After the first phase of the reduction, the return of the 830 nm band corresponding to oxidation of the copper was observed with a half-life of 100 microseconds. Concomitantly, the absorption at 605 and 445 nm due to the reduction of heme a increased. The rates of oxidation of the copper were identical to those of the reduction of heme a and independent of the oxidase concentration. This suggests that the MNA radical reacts with visible copper of the oxidase with a second order rate constant of 1.5 X 10(9) m-1 s-1 and subsequently the electron flows to heme a by intramolecular electron migration with a first order rate constant of 1.8 X 10(4) s-1. An activation energy of the intramolecular electron transfer was calculated to be 2.8 kcal/mol in the range 4-33 degrees C.
Highlights
The reduction of bovine heart cytochrome oxidase by the 1-methylnicotinamide (MNA) radical was investigated by the use of pulse radiolysis
In the presence of 1 mM MNA, a transient spectrum with an absorption maximum at 400 nm was observed at 100 ns after pulse radiolysis. This spectrum is identical to that obtained upon pulse radiolysis of MNA free in aqueous solution [29]
In theabsence of the oxidase, the life time of the MNA radical is longer than 6 ms [29],whereas it decreasedwith the increase of the concentration of the oxidase under the condition of [oxidase] >> [MNA radical]
Summary
From the Instituteof Scientific and Industrial Research, Osaka University, 8-1 Mihogaoka, Ibaraki, Osaka 567, Japan. The reduction of bovine heart cytochrome oxidase by the 1-methylnicotinamide (MNA) radical was investigated by the use of pulse radiolysis. Some of the advantages of the pulse radiolysis technique life of 5 p s This absorption changoebeyed pseudo-first for determining the spectral and kinetic behavior of oneorder kinetics and its rate constainnctreased with the electron reduction products of hemoproteins and flavoproconcentration of the oxidase. This suggests that the teins have been demonstrated [17] This technique is absorption changeat 830 nm is followed by a bimolec- useful for investigating electron transfer reactions in protein ular reaction of the MNA radical with visible copper of the oxidase. We have performed the reactions of the radical other than e, with bovine heart cytochrome oxidase by the use of pulse radiolysis.
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