The role of putidaredoxin and P450 cam in methylene hydroxylation.

Putidaredoxin, the iron sulfur protein from the P450,,,mediated methylene hydroxylase system of Pseudomonas putida, is shown to be essential both as an electron transfer agent and as an effector of P450,,, necessary for product formation. P450,,, is recognized to progress through four well defined states in an ordered reaction cycle of oxidized, and, with substrate, oxidized, reduced, and oxygenated. Combination of reduced putidaredoxin with each of these states of P450,,,, or of oxidized putidaredoxin with the reduced or oxygenated P450,,, in the presence of substrate and 0 2 yields product stoichiometrically. Putidaredoxin is not replaced efficiently by other iron sulfur proteins, nor by the phospholipid from the hepatic microsomal P450 system. Rapid scan spectroscopy shows the oxygenated form of P450,m to be dominant during NADH turnover in the reconstituted system. A scheme is developed for the reaction cycle in which aggregates of the enzyme components are postulated to facilitate electron transfer and determine the nature of the product formed. Analytical treatment of steady state kinetic data suggests that a 1: 1: 1 equivalence of reductase to putidaredoxin to P450,,, is functional and probably reflects participation of 1 molecule each of putidaredoxin, P450,,,,, and possibly reductase in the catalytic complex.