Roles of the axial push effect in cytochrome P450cam studied with the site-directed mutagenesis at the heme proximal site.
To examine the roles of the axial thiolate in cytochrome P450-catalyzed reactions, a mutant of cytochrome P450cam, L358P, was prepared to remove one of the conserved amide protons that are proposed to neutralize the negative charge of the thiolate sulfur. The increased push effect of the thiolate in L358P was evidenced by the reduced reduction potential of the heme. The 15N-NMR and resonance Raman spectra of the mutant in the ferric-CN and in the ferrous-CO forms, respectively, also supported the increased push effect. The maintenance of stereo- and regioselectivities for d-camphor hydroxylation by the mutant suggests the minimum structural change at the distal site. The heterolysis/homolysis ratios of cumene hydroperoxide were the same for wild-type and L358P. However, we observed the enhanced monooxygenations of the unnatural substrates using dioxygen and electrons supplied from the reconstituted system, which indicate the significant role of the push effect in dioxygen activation. We interpret that the enhanced push effect inhibits the protonation of the inner oxygen atom and/or promotes the protonation of the outer oxygen atom in the putative iron-hydroperoxo intermediate (Fe3+ -O-OH) of P450cam. This work is the first experimental indication of the significance of the axial cysteine for the P450 reactivity.
Yoshioka
S
S
Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Japan.
Takahashi
S
S
Ishimori
K
K
Morishima
I
I
D001665:Binding Sites; D002164:Camphor / Q000378:metabolism; D019475:Camphor 5-Monooxygenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D004583:Electrons; D004926:Escherichia coli / Q000201:enzymology; D005292:Ferricyanides / Q000737:chemistry; D005296:Ferrous Compounds / Q000737:chemistry; D006418:Heme / Q000378:metabolism*; D006900:Hydroxylation; D009682:Magnetic Resonance Spectroscopy; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D010957:Plasmids / Q000737:chemistry; D011487:Protein Conformation; D011522:Protons; D013053:Spectrophotometry; D013455:Sulfur / Q000737:chemistry / Q000378:metabolism
D005292:Ferricyanides; D005296:Ferrous Compounds; D011522:Protons; D006418:Heme; D013455:Sulfur; D002164:Camphor; D019475:Camphor 5-Monooxygenase; D003545:Cysteine; D010100:Oxygen