Resonance Raman study on mutant cytochrome P-450 obtained by site-directed mutagenesis

Abstract

Resonance Raman spectra were observed for the threonine-301 to serine or valine mutant as well as the wild type of rabbit liver microsomal cytochrome P-450 [laurate(ω — 1)-hydroxylase] ( P-450(ω — 1), which were prepared through site-directed mutagenesis. The high-spin marker resonance Raman (RR) bands became similarly stronger for all the P-450s examined in the oxidized form upon addition of laurate, and the RR spectra in the higher frequency region of the oxidized, reduced and CO-adduct forms did not distinctly differ among the P-450s examined. Nevertheless, the Fe CO stretching mode ( ν Fe CO ) of the CO adduct exhibited an upshift for the valine mutant, suggesting positional proximity of Thr-301 to bound CO like Thr-252 of P-450 cam, in agreement with the expectation from the sequence analysis. The ν Fe CO band was shifted to higher frequency upon binding of normal alkyl fatty acids with C 10 or longer alkyl chain but little affected by binding of shorter fatty acids.