AUTOXIDATION OF SOLUBLE TRYPSIN-CLEAVED MICROSOMAL FERROCYTOCHROME B-5 AND FORMATION OF SUPEROXIDE RADICALS

The rate and mechanism of autoxidation of soluble ferrocytochrome b5, prepared from liver microsomal suspensions, appear to reflect an intrinsic property of membrane-bound cytochrome b5. The first-order rate constant for autoxidation of trypsin-cleaved ferrocytochrome b5, prepared by reduction with dithionite, was 2.00 .times. 10-3 .+-. 0.19 .times. 10-3 s-1 (mean .+-. SE of the mean, n = 8) when measured at 30.degree. C in 10 mM-phosphate buffer, pH 7.4. At 37.degree. C in aerated 10 mM-phosphate buffer (pH 7.4)/0.15 M-KCl, the rate constant was 5.6 .times. 10-3 s-1. The autoxidation reaction was faster at lower pH values and at high ionic strengths. Unlike ferromyoglobin, the autoxidation reaction of which is maximal at low O2 concentrations, autoxidation of ferrocytochrome b5 showed a simple O2-dependence with an apparent Km for O2 of 2.28 .times. 10-4 M (approx. 20 kPa [kilo Pascals] or 150 mmHg). During autoxidation, 0.25 mol of O2 was consumed/mol of cytochrome oxidized. Cyanide, nucleophilic anions, EDTA and bovine liver catalase [EC 1.11.1.6] each had little or no effect on autoxidation rates. Adrenaline significantly enhanced autoxidation rates, causing a 10-fold increase at 0.6 mM. Ferrocytochrome b5 reduced an excess of cytochrome c in a biphasic manner. An initial rapid phase, independent of O2 concentration, was unaffected by superoxide dismutase. A subsequent slower phase, which continued for up to 60 min, was retarded at low O2 concentrations and inhibited by 65% by [bovine] superoxide dismutase [EC 1.15.1.1] at a concentration of 3 .mu.g/ml. Autoxidation is responsible for a significant proportion of electron flow between cytochrome b5 and O2 in liver endoplasmic membranes, this reaction being capable of generating superoxide anions. A biological role for the reaction is discussed.