J. Biochem, 1984, Vol. 96, No. 5 1491-1500
© 1984 Japanese Biochemical Society
research-article |
Hemoprotein H-450 Identified as a Form of Cytochrome P-450 Having an Endogenous Ligand at the 6th Coordination Position of the Heme
*Department of Biology, Faculty of Science, Kyushu University Higashi-ku, Fukuoka, Fukuoka 812
**Department of Community Health Science, Saga Medical School Nabeshima, Saga, Saga 840-01
***Faculty of Pharmaceutical Sciences, Mukogawa University Nishinomiya, Hyogo 663
****Department of Environmental Sciences, Faculty of Integrated Arts and Sciences, Hiroshima University Hiroshima, Hiroshima 730
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 ß 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, ß, and 
peaks at 448, 540, and 571 nm, whereas the acid form showed Soret, ß, and 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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