Abstract
The mechanism for the reduction of ferric cytochrome P450cam by reduced putidaredoxin, the physiological electron donor for the cytochrome, has been studied by using site-directed mutants of cytochrome P450cam, in which Arg112, an amino acid residue at the presumed binding site for putidaredoxin, was changed to several other amino acid residues. The affinity of reduced putidaredoxin for ferric cytochrome P450cam to form a diprotein complex was decreased greatly by changing Arg112 to a neutral amino acid such as Cys, Met, or Tyr. The rate of intracomplex electron transfer from putidaredoxin to cytochrome P450cam also diminished upon replacing the basic residue with neutral ones, being 42, 18, 4.0, 1.3, and 0. 16 s-1 for Arg (wild type), Lys, Cys, Met, and Tyr enzymes, respectively. Furthermore, the oxidation-reduction potential of cytochrome P450cam (Fe3+/Fe2+ couple) decreased in a similar way to the decrease in the rate of electron transfer upon amino acid substitution; the values were -138, -162, -182, -200, and -195 mV for Arg (wild type), Lys, Cys, Met, and Tyr enzymes, respectively. These results indicate that the amino acid substitution at position 112 affects the oxidation-reduction potential of the heme iron in cytochrome P450cam, thereby diminishing the rate of electron transfer between the two metal centers. The rate of electron transfer from putidaredoxin to oxyferrous cytochrome P450cam also diminished upon substitution of Arg112 with a neutral amino acid.
Highlights
The mechanism for the reduction of ferric cytochrome P450cam by reduced putidaredoxin, the physiological electron donor for the cytochrome, has been studied by using site-directed mutants of cytochrome P450cam, in which Arg112, an amino acid residue at the presumed binding site for putidaredoxin, was changed to several other amino acid residues
The oxidation-reduction potential of cytochrome P450cam (Fe3؉/ Fe2؉ couple) decreased in a similar way to the decrease in the rate of electron transfer upon amino acid substitution; the values were ؊138, ؊162, ؊182, ؊200, and ؊195 mV for Arg, Lys, Cys, Met, and Tyr enzymes, respectively. These results indicate that the amino acid substitution at position 112 affects the oxidation-reduction potential of the heme iron in cytochrome P450cam, thereby diminishing the rate of electron transfer between the two metal centers
We found in this study that charge neutralization at Arg112, a surface amino acid residue of P450cam, affected the redox potential of the heme iron, which is buried in the interior of the protein; the redox potential of the wild-type P450cam, Ϫ138 mV, was lowered to Ϫ182 to Ϫ200 mV in the mutant enzymes having a neutral amino acid residue such as Cys, Met, or Tyr at the 112-position
Summary
P450cam, cytochrome P450 (CYP101) isolated from Pseudomonas putida, which catalyzes the conversion of d-camphor to 5-exo-hydroxycamphor; Oxy-P450cam, a ferrous oxygenated form of cytochrome P450cam; Pd, putidaredoxin; PdR, putidaredoxin reductase. We further elucidate the role of Arg112 in the electron transfer reaction from reduced Pd to ferric P450cam by using site-directed mutants of P450cam, in which the Arg residue was changed to a Lys, Cys, Tyr, or Met residue. Since they have different charges and hydrogen-bonding capacities with one another, we had hoped that their use might give us a clue to identify the role of the basic residue Arg112 in the Pd-P450cam interaction. The cationic charge at Arg112 was found to be important in the second electron transfer, i.e. in the reduction of the ferrous oxygenated form of P450cam (oxy-P450cam) by reduced Pd
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