Camphor binding by Pseudomonas putida cytochrome P-450

Abstract

In this report, a procedure is described for the large-scale purification from the bacterium Pseudomonas putida of two components of the camphor methylene hydroxylase system, cytochrome P-450 and putidaredoxin. The absorption spectra and circular dichroism spectra of oxidized, reduced, and the carbon monoxide-complex forms of cytochrome P-450 in the presence and absence of camphor are shown. The heme iron of cytochrome P-450 is in a high-spin state with five unpaired electrons in the presence of camphor and a low-spin state with one unpaired electron in the absence of camphor as determined by magnetic susceptibility and electron paramagnetic resonance measurements, respectively. Cations are shown to potentiate camphor binding by cytochrome P-450; in the presence of 0.1 m KCl, the association constant of camphor and cytochrome P-450 is 0.47 ± 0.07 × 10 6 m −1. A possible mode of interaction of camphor, cations, and cytochrome P-450 is discussed. The specific activity of cytochrome P-450, as measured by the rate of oxygen utilization in the presence of an excess of the other components of the camphor methylene hydroxylase system, is 600 nmoles/min/nmole of cytochrome P-450. Anaerobic titration of cytochrome P-450 with Na 2S 2O 4 indicates that cytochrome P-450 is a one-electron acceptor. A possible reaction mechanism of cytochrome P-450 in the interaction of substrate and oxygen in the hydroxylation reaction is presented.