Hemoprotein H-450 Identified as a Form of Cytochrome P-450 Having an Endogenous Ligand at the 6th Coordination Position of the Heme

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Abstract
Hemoprotein H-450 was purified from rat liver cytosol to homogeneity by an improved procedure. The purified H-450 showed a subunit molecular weight of 64,000 daltons and contained 0.7–0.9 mol of protoheme per mol subunit. Among rat tissues examined, liver and kidney contained significant amounts of H-450 in the cytosol. Oxidized H-450 showed a Soret peak at 428 nm and a broad β band at around 550 nm. Reduced H-450 was found to exist in two interconvertible forms, alkaline and acid forms. The alkaline form showed Soret, β, and α peaks at 448, 540, and 571 nm, whereas the acid form showed Soret, β, and α peaks at 425, 530, and 558 nm. The spectral properties of both oxidized and reduced H-450 in alkaline medium resemble those of cytochrome P-450 having a nitrogenous ligand at the 6th coordination position of the heme. Upon addition of low concentrations of HgCl2, H-450 was converted to a denatured form both in the oxidized and the reduced states and lost its unique spectral characteristics. Addition of carbon monoxide to reduced H-450 produced a new spectral species which resembled that of the reduced carbon monoxide complex of P-420, a denatured form of cytochrome P-450. Comparison of the EPR signal of oxidized H-450 with those of a cytochrome P-450, P-450(PB-1), and several model compounds indicated the presence of a thiolate anion at the 5th coordination position of the heme of H-450. Judging from EPR data, oxidized H-450 also converts between acid and alkaline forms, whose signals were observed at g = 1.867, 2.31, and 2.507 and at g = 1.910, 2.28, and 2.424, respectively. These lines of evidence indicate that the 5th and 6th coordination positions of the heme of H-450 are a thiolate and a nitrogenous group, respectively. With respect to the heme environments, H-450 is a member of the cytochrome P-450 family, and has a nitrogenous ligand at the 6th coordination position of the heme.

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