P-450 PB-4 Research Articles

Circular dichroism spectra of purified cytochromes P-450 from rabbit liver microsomes

Circular dichroism (CD) spectra were measured for cytochromes P-450 ( P-450) purified from phenobarbital- and 3-methylcholanthrene-induced rabbit liver microsomes. No striking difference in α-helix content was seen between phenobarbital-induced P-450 (PB P-450) (50%), phenobarbital-induced P-448 (PB P-448) (40%) and 3-methylcholanthrene-induced P-448 (MC P-448) (45–50%) in terms of ultraviolet CD spectra. Strong negative CD spectra associated with 3-methylcholanthrene transitions for MC P-448 in the near-ultraviolet region (250–310 nm) and weaker negative CD spectra associated with Soret transitions for PB P-448 ([ φ] = 50 000) and MC P-448 ([ φ] = 160 000), indicated that structures of these preparations are strikingly different from each other. Reduction of P-450 and P-448 led to a remarkable decrease of the Soret CD trough, suggesting that reduction was accompanied by a striking conformational change in the vicinity of the heme. Since CO complexes of reduced P-450 and P-448 showed a CD trough and an S-shaped CD, respectively, associated with the absorption peak at 450 nm, the heme vicinities are remarkably different from each other. The CD spectra in the visible region are also discussed. It was noticed that P-420, the denatured form of P-450, exhibited no CD spectra in the Soret and visible regions.

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.

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 three 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 iron dpi orbital to the porphyrin II (eg) orbital and, consequently, the strong pi basicity of the fifth 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 hemoglobin 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, whereas 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 two P-448 preparations in the reduced state. The reduced form of cytochrome P-420, produced by laser illumination, exhibits two sets of Raman lines and, therefore, seems to be a mixture of both high- and low-spin species.