Pseudomonas putida cytochrome P-450: Characterization of an oxygenated form of the hemoprotein

Abstract

When ferrous cytochrome P-450, purified from Pseudomonas putida, was mixed with oxygen in the presence of its organic substrate, d-camphor, a new spectrally distinct species was observed whose absorption spectrum had maxima at 355, 418 and 555 nm. The absorption spectrum of this new species was essentially unchanged by cooling the reaction mixture to the temperature of liquid nitrogen. The new spectral species is rather stable in the absence of the iron-sulfur protein, putidaredoxin, a physiological electron donor for cytochrome P-450 and can be reversibly transformed into carbon monoxy-cytochrome P-450 by varying the relative partial pressures of oxygen and carbon monoxide. No electron paramagnetic resonance signal attributable to the new species could be detected between 500 and 4000 gauss at either 10.5 or 77 ° K. Oxygen, camphor and the ferrous cytochrome are required for the formation of the new spectral species. The reaction is first order with respect to both oxygen and cytochrome P-450 concentrations and the second order reaction rate constant is 7.7 × 10 5 m −1 sec −1 at 4 °. The formation of the new species proceeded without detectable intermediates. The dissociation of oxygen from the oxygenated form is first order with a rate constant of 1.1 sec −1 at 4 °. The new spectral species undergoes slow decomposition to the ferric form of the hemoprotein and is most stable in the pH range 7–8 and at temperatures below 10 °. The decomposition of the ferrous oxygen complex to the ferric form is first order with respect to the oxygenated species (rate constant = 10 −4 sec −1 at pH 7.4 and 4 °) and also proceeds without detectable intermediates. The rate of transformation to the ferric form is increased by increasing the hydrogen ion concentration, raising the temperature, or increasing the ionic strength of the reaction medium. Based on these findings, the new spectral species is interpreted to be an oxygenated form of cytochrome P-450, presumably a ternary complex of oxygen, camphor, and ferrous cytochrome P-450.