Modulation of redox potential and alteration in reactivity via the peroxide shunt pathway by mutation of cytochrome P450 around the proximal heme ligand.
In the thermophilic cytochrome P450 from the thermoacidophilic crenarchaeon Sulfolobus tokodaii strain 7 (P450st), a phenylalanine residue at position 310 and an alanine residue at position 320 are located close to the heme thiolate ligand, Cys317. Single site-directed mutants F310A and A320Q and double mutant F310A/A320Q have been constructed. All mutant enzymes as well as wild-type (WT) P450st were expressed at high levels. The substitution of F310 with Ala and of A320 with Gln induced shifts in redox potential and blue shifts in Soret absorption of ferrous-CO forms, while spectral characterization showed that in the resting state, the mutants almost retained the structural integrity of the active site. The redox potential of the heme varied as follows: -481 mV (WT), -477 mV (A320Q), -453 mV (F310A), and -450 mV (F310A/A320Q). The trend in the Soret band of the ferrous-CO form was as follows: 450 nm (WT) < 449 nm (A320Q) < 446 nm (F310A) < 444 nm (F310A/A320Q). These results established that the reduction potential and electron density on the heme iron are modulated by the Phe310 and Ala320 residues in P450st. The electron density on the heme decreases in the following order: WT > A320Q > F310A > F310A/A320Q. The electron density on the heme iron infers an essential role in P450 activity. The decrease in electron density interferes with the formation of a high-valent oxo-ferryl species called Compound I. However, steady-state turnover rates of styrene epoxidation with H2O2 show the following trend: WT approximately equal to A320Q < F310A approximately equal to F310A/A320Q. The shunt pathway which can provide the two electrons and oxygen required for a P450 reaction instead of NAD(P)H and dioxygen can rule out the first and second heme reduction in the catalytic process. Because the electron density on the heme iron might be deeply involved in the k cat values in this system, the intermediate Compound 0 which is the precursor species of Compound I mainly appears to participate dominantly in epoxidation with H2O2.
Matsumura
Hirotoshi
H
Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan.
Wakatabi
Masahiro
M
Omi
Sayaka
S
Ohtaki
Akashi
A
Nakamura
Nobuhumi
N
Yohda
Masafumi
M
Ohno
Hiroyuki
H
D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D015870:Gene Expression; D006418:Heme / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D009154:Mutation / Q000235:genetics; D010084:Oxidation-Reduction; D010545:Peroxides / Q000378:metabolism*; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D020058:Styrene / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D008024:Ligands; D010545:Peroxides; D006418:Heme; D020058:Styrene; D003577:Cytochrome P-450 Enzyme System