Resonance raman studies of hepatic microsomal cytochromes P-450: evidence for strong .pi. basicity of the fifth ligand in the reduced and carbonyl complex forms

Abstract

Resonance Raman spectra have been measured for cytochromes P-450 purified from liver microsomes of phenobarbital-treated rabbits (PB P-450 and PB P-448) and of 3-methylcholanthrene-treated rabbits (MC P-448). In the reduced state, all 3 cytochromes P-450 exhibit Raman spectra of ferrous high-spin type but show the so-called oxidation state marker (band IV) at unusually low frequencies, indicating extensive delocalization of electrons from the Fe d.pi. orbital to the porphyrin II*(eg) orbital and, consequently, the strong .pi. basicity of the 5th ligand of the heme iron. The reduced CO complexes of the cytochromes P-450 also exhibit band IV at markedly lower frequencies than CO complexes of Hb and myoglobin. These anomalies observed for the reduced form and CO complex disappear upon conversion of the cytochromes to the catalytically inactive form called cytochrome P-420. Oxidized PB P-450 shows a Raman spectrum which is characteristic of typical ferric low-spin heme compounds, but those of PB P-448 and MC P-448 are of the ferric high-spin type. PB P-450 is also clearly distinguishable from the 2 P-448 preparations in the reduced state. The reduced form of cytochrome P-420, produced by laser illumination, exhibits 2 sets of Raman lines and seems to be a mixture of both high- and low-spin species.