Mitochondrial Monoamine Oxidase

As an approach to the investigation of inhibiting effects of substituted hydrazines with monoamine oxidases, highly active bovine kidney mitochondrial oxidase was used to study the mechanism of phenylhydrazine-induced inactivation. Treatment of the enzyme under N2 with a 3-fold excess of inhibitor produced almost complete reduction of the enzyme flavin. Introduction of oxygen then caused 80% reoxidation of flavin with equivalent restoration of enzyme activity. With oxygen present initially phenylhydrazine effected almost complete irreversible inhibition. It appears that the enzyme catalyzed the oxidation of phenylhydrazine producing a highly unstable product, phenyldiazene (phenyldiimide), which inactivated the enzyme irreversibly. Such an inactivation was observed with phenyldiazene itself, prepared in situ via the decarboxylation of phenylazoformate. Our evidence, including a Hammett study correlating the effects of nuclear substitution in phenylhydrazines with rates of hydrazine-induced inhibition (ρ = -1.9) suggests that initial phenylhydrazine reduction of the flavoenzyme is rate-determining in the over-all (aerobic) inhibition process. Inactivation of the enzyme in air by treatment with either phenylhydrazine or phenyldiazene generated a damaged reduced type flavin, stable to autoxidation. Use of [1-14C]phenylhydrazine with the oxidase gave a stable 14C-protein product (1.4 residues of inhibitor bound per enzyme subunit). It is concluded that enzyme inactivation involves the formation of a stable flavin-inhibitor adduct, apparently without involvement of enzyme sulfhydryl groups. Similar effects were observed on treatment of the enzyme with 2-phenylethylhydrazine and dl-1-methyl-2-phenylethylhydrazine, suggesting that inhibition of the oxidase with aralkylhydrazines also involves diazene intermediates. Phenylethylhydrazine, however, was extensively converted to phenylacetaldehyde hydrazone (phenylethylidene hydrazine) either through rearrangement of the derived diazene or less probably through oxidation by an alternate route. The hydrazone was inactive as an enzyme inhibitor. Thus, the parent hydrazine had produced only 5% of the inhibition effected by an equimolar quantity of phenylhydrazine or alternatively by 1-methyl-2-phenylethylhydrazine, where the 1-methyl group presumably blocked hydrazone formation. Benzylhydrazine, as inhibitor only slightly less effective than phenylhydrazine, was minimally oxidized to hydrazone.