Studies of the oxygen binding site of cytochrome P-450. Nitric oxide as a spin-label probe.

Pseudomonas putida and rat liver microsomal cytochromes P-450 form both ferric. and ferrous.NO complexes. The ferric.NO complexes are stable and do not possess ESR spectra in the temperature range 5-300 K which suggests that the unpaired electron of the bound NO is spin-paired with the single unpaired d electron of the hemin iron. The ferrous.NO complex of the bacterial cytochrome is quite stable; however, a portion of the comparable complex of the microsomal enzyme rapidly denatures to a five-coordinate P-420.NO species. The percentage of the ferrous.NO complex of the microsomal enzyme which becomes five-coordinate appears to be dependent upon the amount of high spin cytochrome present in the original ferric enzyme since it is less in the presence of exogenous Type I substrates. The addition of the substrate, d-camphor, to the bacterial cytochrome P-450 does not affect the optical absorbance or electron spin resonance (ESR) spectra of either the ferric. or ferrous.NO complexes. The optical absorbance difference spectrum of the ferricNO uersus ferric liver microsomal cytochrome P-450 is dependent upon the percentage of the enzyme in the high (substrate-bound) uersus low spin (substrate-free) state. The Soret absorbance band maxima of the ferric. and ferrous.NO complexes of these cytochromes P450 are shifted to longer wavelengths than those of the corresponding complexes of horseradish peroxidase. The ESR spectra of the ferrous.NO complexes of cytochromes P-450 reveal rhombic symmetry with a triplet hyperfine interaction in the g, signal. Unlike horseradish peroxidase, there is no nine-line superhyperfine structure for the g,