Influence of Oxygen on the Radiochemical Inactivation of the Microsomal Monooxygenase Complex

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Abstract
The effect of γ-ray irradiation on benzo(a)pyrene (BP) monooxygenation by microsomes from 3-methylcholanthrene-induced mouse livers, was investigated both in the presence and in the absence of oxygen in phosphate buffer. Anaerobically irradiated microsomal suspensions showed an exponentially dose-dependent inhibition of the hydroxylase activity. The active site of the P-448 cytochrome was unaltered in regard to substrate binding and to the height of the 448 nm absorption band of the reduced haeme-carbon monoxide complex. The whole inactivation process is tentatively explained in terms of radical-induced protein-protein cross-links at the surface of the microsomes. Oxygen abolished this reaction and inactivated the microsomal monooxygenase system through a route entirely ruled by the peroxidation of membrane phospholipids. The NADPH-cytochrome c reductase and epoxide hydrolase activities were only slightly affected, but cytochrome P-448 was damaged following a complicated process. In fact, irradiation in the presence of O2 brought out two functionally and spectrally distinct subgroups of cytochrome P-448 referred to as (A) and (B) and including 40 per cent and 60 per cent of the whole P-448 content, respectively. The (A)-type cytochrome was inactive in BP metabolization but appeared fully radioresistant after a 2700 Gy irradiation. It utilized the lipid peroxides for the transformation of some substrate found in the microsomal membrane into a ligand X which conferred on the ferrous haeme an absorption band at 449 nm similar to that for a Fe(II)-CO complex. Lipid peroxides produced upon warming at 37°C in non-irradiated microsomes had the same effect. The exchange of X for CO in the reduced state was characterized by laser flash-photolysis. In contrast, CO was required for the 448 nm absorption to appear in the reduced (B)-type cytochrome. This P-448 cytochrome turned into a P-420 form inactive in BP-monooxygenation upon prolonged radiolysis. The P-448→P-420 evolution showed a pronounced lag-time parallel with the completion of lipid peroxidation in the outer layer of the microsomal membrane. The irreversible formation of P-420 did not result from attack of the P-448 active site by the lipid hydroperoxides. Alteration of the membrane-cytochrome P-448 interaction subsequent to lipid peroxidation is proposed as a structural model thereof. The reaction was unchanged upon substitution of rat liver for mouse liver microsomes.