Complete amino acid sequence of a cytochrome P-450 isolated from beta-naphthoflavone-induced rabbit liver microsomes. Comparison with phenobarbital-induced and constitutive isozymes and identification of invariant residues.

Abstract

The complete covalent structure of a cytochrome P-450, form 4, isolated from liver microsomes of beta-naphthoflavone-induced rabbits was determined. The S-carboxyamidomethylated protein was cleaved with cyanogen bromide, endoproteinase Lys-C, and trypsin before and after succinylation. Selected peptides from CNBr digests of alkylated rabbit cytochrome P-450 forms 3a and 3c were also isolated and sequenced. Form 4 exhibited microheterogeneity due to the presence of several truncated forms. The existence of multiple NH2-terminal residues for form 4 was confirmed by the isolation and sequence analysis of the corresponding tryptic peptides. The predominant form contained 514 residues, corresponding to Mr 58,030. A peptide having Gly-232 and Gln-246 replaced by Ser and Asn residues, respectively, was also found in the isozyme preparation investigated here. The amino acid sequences of form 4 and selected peptide sequences from forms 3a and 3c were compared with the primary structures of forms 2 and 3b (previously determined in this laboratory). This comparison identified some 90 invariant residues. A cysteinyl residue at position 456, earlier reported as the heme-binding cysteine 436 (Heineman, F. S., and Ozols, J. (1982) J. Biol. Chem. 257, 14988-14999), was also present in forms 4, 3a, and 3c. Other single invariant residues identified were form 4/forms 2,3b, Trp-132/121, and His 270/252. The tyrosyl residues at positions 71/62 and 365/348 were also invariant. The latter is present in the "conserved segment" of the protein, residues 363/346 to 375/359, and may be involved in the substrate binding of cytochrome P-450. Also a lysyl residue, formerly identified by other laboratories to be involved in the electron transfer between the reductase and cytochrome P-450 form 2, was invariant in all five species. This lysyl residue corresponds to Lys-402 in form 4 or Lys-384 in the other forms.