Methylene Oxidation of Alkyl Sulfates by Cytochrome P450 and a Role for Conformational Selection in Substrate Recognition.

Cytochrome P450 (P450) is a flavoprotein reductase-heme fusion protein from the bacterium that has been well-characterized in many biophysical aspects. Although the enzyme is known to catalyze the hydroxylation of medium and long-chain fatty acids at high rates, no definitive physiological function has been associated with this process in the organism other than a possible protective role. We found that P450 rapidly hydroxylates alkyl sulfates, particularly those with 12-16 carbons (i.e., including dodecyl sulfate) in a similar manner to the fatty acids. The products were characterized as primarily -1 hydroxylated alkyl sulfates (plus some -2 and -3 hydroxylation products), and some further oxidation to dihydroxy and keto derivatives also occurred. Binding of the alkyl sulfates to P450 converted the iron from the low-spin to high-spin form in a saturable manner, consistent with the catalytic results. Rates of binding decreased as a function of increasing concentration of dodecyl sulfate or the fatty acid myristate. This pattern is consistent with a binding model involving multiple events and with conformational selection (equilibrium of the unbound enzyme prior to binding) instead of an induced fit mechanism. Neither C-H bond-breaking nor product release was found to be rate-limiting in the oxidation of lauric acid. The conformational selection results rationalize some known crystal structures of P450 and can help explain the flexibility of P450 and engineered forms in accepting a great variety of substrates.