Alteration of high and low spin equilibrium by a single mutation of amino acid 209 in mouse cytochromes P450.
The identities of the amino acid at position 209 are most critical in determining specific coumarin 7- and steroid 15 alpha-hydroxylase activity in P450coh and P450(15)alpha, respectively. This system, therefore, provides us with an excellent model to study the structural basis for P450 specificity as a monooxygenase. We expressed in Saccharomyces cerevisiae a series of the mutated P450s in which residue 209 was substituted with the various amino acids and characterized the spectral property and hydroxylase activity of these mutated P450s. The positioning of a hydrophobic residue including Phe, Leu, and Val at position 209 resulted in shifting the P450 to the high-spin state, while a charged amino acid such as Lys or Asp produced the low-spin form. Moreover, a P450 with Asn or Gly in this position exhibited spectra indicating a mixture of the high- and low-spin forms. This spin alteration, depending upon the hydrophobicity and size of residue at position 209, indicates that this position is likely to reside close to the sixth axial ligand on the distal surface of the heme in these P450s. This proximity of residue 209 to the ligand may explain the critical role of this residue in determining the hydroxylase specificity and activity of these P450s.
Iwasaki
M
M
Pharmacogenetics Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709.
Juvonen
R
R
Lindberg
R
R
Negishi
M
M
D000596:Amino Acids / Q000235:genetics*; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002384:Catalysis; D065637:Cytochrome P-450 CYP2A6; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D051379:Mice; D006899:Mixed Function Oxygenases / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet; D013250:Steroid Hydroxylases / Q000235:genetics*
D000596:Amino Acids; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D065637:Cytochrome P-450 CYP2A6; C050356:steroid 15-alpha-hydroxylase