Resonance raman spectra of bovine adrenal cytochrome P-450scc

Abstract

Resonance Raman spectra were observed for a mitochondria-type cytochrome $\text{P-450}$ ($\text{P-450}{}_{\mathrm{<mtext>SCC</mtext>}}$ for the first time. Reduced $\text{P-450}{}_{\mathrm{<mtext>SCC</mtext>}}$ at pH 7.4 exhibited the $\text{v}{}_4$ line at 1342 cm⁻¹, which is an unusually low frequency compared with an ordinary protohemoprotein but is common to the family of cytochrome $\text{P-450}$, suggesting the coordination of a strong π-donor such as thiolate anion at the fifth coordination position of the heme iron. The anomaly was preserved for the CO-complex of the reduced form. The $\text{v}{}_{10}$ line of oxidized $\text{P-450}{}_{\mathrm{<mtext>SCC</mtext>}}$ with a substrate was observed at 1617 cm⁻¹. This frequency and those of other structure-sensitive bands implied that the heme iron of oxidized $\text{P-450}{}_{\mathrm{<mtext>SCC</mtext>}}$ adopts the hexa-coordinate high-spin structure, in contrast with the high-spin type cytochrome $\text{P-450}$ purified from phenobarbital- or 3-methylcholanthrene-treated rabbit liver microsomes which presumably have a penta-coordinate structure. In the presence of 20α-hydroxycholestero, oxidized $\text{P-450}{}_{\mathrm{<mtext>SCC</mtext>}}$ gave the $\text{v}{}_{10}$ line at 1637 cm⁻¹, i.e., at a frequency similar to that of low-spin type cytochrome $\text{P-450}$. The alkaline-denatured $\text{P-450}{}_{\mathrm{<mtext>SCC</mtext>}}$ preparation in the presence of both dithiothreitol and EDTA, but not the $\text{P-450}{}_{\mathrm{<mtext>SCC</mtext>}}$ gave the $\text{v}{}_{10}$ line at 1637 cm⁻¹, i.e., at a frequency similar to that of low-spin type cytochrome $\text{P-450}$. The alkaline-denatured $\text{P-420}{}_{\mathrm{<mtext>SCC</mtext>}}$ preparation in the presence of both dithiothreitol and EDTA, but not the $\text{P-450}{}_{\mathrm{<mtext>SCC</mtext>}}$.