A Role of the Putidaredoxin COOH-terminus in P -450 cam (Cytochrome m ) Hydroxylations

Abstract

Methylene hydroxylation by cytochrome P-450(cam) (cytochrome m) can be resolved into four distinct steps: substrate addition, m(o) --> m(os); reduction, m(os) --> m(rs); dioxygen addition, m(rs) --> m(O2) (rs); followed by a second putidaredoxin (Pseudomonas putida ferredoxin)-mediated reduction and product formation. The isolated ferrous oxy-substrate complex exhibits first-order decay kinetics with the relatively slow rate constant of k [unk] 0.01 sec(-1), at 25 degrees , without product release. Putidaredoxin addition accelerates the decomposition with second-order kinetics, k [unk] 51,000 M(-1) sec(-1), and initiation of product formation. Cytochrome m forms a complex with putidaredoxin with dissociation constant of K(D) = 3 muM. In the complete three-protein hydroxylase system, consisting of cytochrome m, putidaredoxin, and the reductase (a DPNH-specific flavo-protein), camphor hydroxylation occurs with a stoichiometry of 1 mole each of DPNH and O(2) used per mole of product formed; the K(M) for putidaredoxin is about 4.2 muM.Putidaredoxin, on treatment with carboxypeptidase A, loses one molecule each of tryptophan and glutamine sequentially from the carboxy terminus to expose a terminal arginine. The tryptophan-free product has been separated from native putidaredoxin and other impurities, and retains the visible and electron paramagnetic resonance spectra and the redox potential of the active center of native putidaredoxin. This modified redoxin binds less tightly to cytochrome m, K(D) [unk] 150 muM, and is 50 times less effective in stimulation of the m(O2) (rs) decay rate. A similar decrease in specific activity is observed in the complete hydroxylase system.