Interaction of 5-bromocamphor with cytochrome P-450 cam. Production of 5-ketocamphor from a mixed spin state hemoprotein.

Abstract

Camphor is stereospecifically hydroxylated by the soil bacterium Pseudomonas putida at the 5-exo position by a cytochrome P-450 mixed function oxidase system consisting of a flavoprotein reductase; putidaredoxin, an iron-sulfur oxidation-reduction transport-effector protein; and the P-450 hemoprotein. We have studied the interaction of a substrate analog of camphor, 5-exo-bromocamphor, with this cytochrome P-450 mixed function oxidase system in order to probe the molecular mechanisms of electron transport and catalytic substrate oxygenation. 5-exo-Bromocamphor was found to bind tightly in a 1:1 complex with P-450 with a dissociation constant of 2.9 microM, very near that for the normal camphor substrate. Contrary to camphor, however, the 5-exo-bromocamphor-bound material exhibits only a 46% population of the high spin form of the macromolecule. Measurement of the oxidation-reduction potential of the bromocamphor-P-450 complex yields a value of E0' = -246 mV, intermediate between that of substrate-free (-300 mV) and camphor-bound (-173 mV) cytochrome. These data are interpreted in terms of a thermodynamic linkage model relating spin, substrate, and oxidation-reduction equilibria in the P-450 monoxygenase system. 5-exo-Bromocamphor is readily metabolized by the P-450cam mixed function oxidase to 5-ketocamphor at rates and yields similar to that of the normal substrate, camphor, suggesting abstraction of the endo-hydrogen of 5-exo-bromocamphor and oxygen addition to produce a transient 5-bromo-5-hydroxycamphor intermediate. Implications of these chemical findings for the chemical mechanisms of P-450-catalyzed oxygenations and the flexibility of substrates at the active site of the macromolecule are discussed.