Mitochondrial cytochrome P-450sec. Mechanism of electron transport by adrenodoxin.

Abstract

Complex formation between adrenodoxin and cytochrome P-450,, has been monitored via the increase in high spin cytochrome.A hyperbolic dependence upon adrenodoxin (Kd = 8 X lo-' M) was changed to a sigmoid dependence by the addition of adrenodoxin reductase.The change is quantitatively explained by the change in the concentration of free adrenodoxin due to formation of a strong adrenodoxin reductase-adrenodoxin complex (Kd = 1.8 X lo-' M) that does not significantly bind P-450.,,.No evidence was obtained for a ternary complex formation involving all three enzymes.Similarly, under steady state conditions, clear evidence was presented that a ternary complex is not a functional intermediate in electron transport.Cholesterol side chain cleavage dependence on adrenodoxin concentration showed an increasingly sigmoid pattern as the concentration of adrenodoxin reductase was increased.These plots could be transformed into Michaelis-Menten functions of free adrenodoxin (K, = 0.4 p ~) by assuming that adrenodoxin formed separate complexes with its reductase and cytochrome P-450.,, and that a ternary complex did not form to any significant degree.Additionally, the present data could be most consistently interpreted, assuming at least a &fold decrease in the affinity of adrenodoxin reductase to adrenodoxin upon reduction of both enzymes.These results indicate that adrenodoxin transports electrons from adrenodoxin reductase to cytochrome P-450 by shuttling between these two enzymes.During cholesterol side chain cleavage at saturating concentrations of adrenodoxin and adrenodoxin reductase, adrenodoxin was fully reduced and the reduction of adrenodoxin was not rate-limiting.At suboptimal concentrations of adrenodoxin reductase, a near steady state level of reduced adrenodoxin was rapidly established.The rate of cholesterol side chain cleavage did not correlate with the concentration of reduced adrenodoxin but instead correlated directly (r = 0.965) with the percentage of adrenodoxin reduced at the midpoint of the reaction.It is inferred that oxidized adrenodoxin inhibits cholesterol side chain cleavage by competition with reduced adrenodoxin for binding to P-45OS,, during the catalytic cycle.The mammalian mitochondrial steroid and sterol monooxygenase systems characterized to date consist of three enzymes: a flavoprotein NADPH ferredoxin reductase, a ferre-