Topological studies of the steroid hydroxylase complexes in bovine adrenocortical mitochondria

The topology of the steroid hydroxylase complexes in bovine adrenocortical mitochondria was studied by using nonpenetrating artificial electron acceptors and the impermeable protein reagent diazobenzenesulfonate. Inhibition of steroid hydroxylase activity by ferricyanide and dichlorophenolindophenol sulfonate was only observed in mitochondria which had been damaged by various techniques. Intact mitochondria were not inhibited by these reagents. The reaction was monitored by oxygen uptake due to hydroxylation of deoxycorticosterone, as well as P-450 reduction and corticosterone formation. The results obtained were similar regardless of how the activity was measured. Labeling of the mitochondria with the nonpenetrating protein reagent diazobenzenesulfonate also inhibited P-450 reduction and corticosterone formation in mitochondria which had been damaged prior to addition of this reagent. Intact mitochondria which were labeled with this reagent showed very little inhibition of both activities. These results strongly suggest that all protein components of the steroid 11beta-hydroxylase system are located on the matrix side of the mitochondrial inner membrane. The inability of ferricyanide, dichlorophenolindophenol sulfonate, and diazobenzenesulfonate to inhibit the malate-dependent reduction of P-450 in intact mitochondria implies that all the P-450-dependent mitochondrial steroid hydroxylase systems are located on the matrix side of the inner mitochondrial membrane.