Site-directed mutageneses of rat liver cytochrome P-450d: axial ligand and heme incorporation

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Abstract
By oligonucleotide-directed mutageneses, 13 substitutions of amino acids at the carboxy-terminal region of rat liver cytochrome P-450d were done as follows: (A) Phe-449 .fwdarw. Tyr; (B) Gly-450 .fwdarw. Ser; (C) Leu-451 .fwdarw. Ser; (D) Gly-452 .fwdarw. Glu; (E) Lys-453 .fwdarw. Glu; (F) Arg-454 .fwdarw. Leu; (G) Arg-455 .fwdarw. Gly; (H) Cys-456 .fwdarw. Tyr; (I) Cys-456 .fwdarw. His; (J) Ile-457 .fwdarw. Ser; (K) Gly-458 .fwdarw. Glu; (L) Glu-459 .fwdarw. Ala; (M) Ile-460 .fwdarw. Ser. The CO-bound reduced forms of the wild type and mutants B-G, J, L, and M gave Soret peaks at 448 nm. The CO complex of mutant A gave a Soret peak at 445 nm. The intensities of the CO-bound forms of mutants A, C, D, and J were very small compared with that of the wild-type complex. The CO-reduced forms of mutants H, I, and K did not give a Soret peak around 450 nm at all. The 448-nm peak of mutant F was unstable and quickly disappeared with the concomitant appearance of a peak at 420 nm. These findings, together with data in the literature, indicate that (1) invariant Cys-456 at the carboxy-terminal region of P-450d is an axial ligand of the heme iron of the eukaryotic P-450''s; (2) hydrophobic amino acids such as Phe-449, Leu-451, Gly-452, and other hydrophobic amino acids such as Ile-457 and Gly-458 next to the axial ligand are apparently very important for the apoprotein to hold and/or incorporate the heme plane at the active site of P-450; and (3) Arg-454 interacts with the heme propionate as His-355 of P-450cam does [Poulos, T. L., Finzel, B. C., Gunsalus, I. C., Wagner, G. C., and Kraut, J. (1985) J. Biol. Chem. 260, 16122-16130].

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