Abstract
To identify amino acids of cytochrome P450d (P450d) which participate in the interaction with NADPH-cytochrome P450 reductase, we changed conserved ionic amino acids of P450d to others by site-directed mutagenesis. Turnover numbers (0.032-0.008 min-1) of purified mutants Lys94-Glu, Lys99-Glu, Lys105-Glu, Lys440-Glu, Lys453-Glu, Arg455-Glu, and Lys463-Glu toward 7-ethoxycoumarin were much lower than that (0.380 min-1) of the wild type at 25 degrees C. Reduction rates (less than 0.054 s-1) of the heme of all mutants (0.1 microM) in the presence of NADPH and the reductase (0.3 microM) were much lower than that (5.9 s-1) of the wild type. Furthermore, a turnover number (0.042 min-1) of a microsomal triple mutant (Arg135-Leu + Arg136-Leu + Arg137-Leu) of a conserved Arg cluster was much lower than that (0.674 min-1) of the wild type at 37 degrees C. Thus, we suggest that Lys94, Lys99, Lys105, Lys440, Lys453, Arg455, Lys463, and perhaps the Arg cluster Arg135-Arg136-Arg137 of P450d will participate in the intermolecular electron transfer process by forming ionic bridges between the two proteins and/or by orienting appropriate geometry for electron transfer on the interfacial surface between the two proteins.
Highlights
Site-directed Mutagenesis hand, a non-heme iron protein, putidaredoxin, and a flavoprotein, NADH-putidaredoxin reductase, are necessary as INTERACTION WITH NADPH-CYTOCHROME P450 REDUCTASE*
From the $Chemical Research Instituteof Non-aqueous Solutions, Tohoku Uniuersity, Katahira, Sendai 980, Japan and the TDepartmenotf Chemistry, Faculty of such as Arg7*,Argil2, Lys344a, nd Arg364have been proposed for the surface residue of P450, involved in theinteraction with putidaredoxin [17, 18].In a similar way, mitochondrial P450s such as P450, require a non-heme iron protein, adrenodoxin, and aflavoprotein, NADPH-adrenodoxin reductase, Science, Tohoku Uniuersity, Aoba, Sendai 980, Japan as electron mediators for catalytic activities (Ref. 19 and references cited therein)
Which participate in the interaction with NADPH-cy- Lys338or L Y S ~(o~r' Lys377o)f P450, has been suggested to be tochrome P450 reductase, we changed conserved ionic involved in the electrostatic interactions with adrenodoxin amino acids of P450d to others by site-directed muta- (20,Zl)
Summary
Expression oPf 450d mutants in yeast was done as described previously [24,25,26,27,28]. Preparations of yeast microsomes and purification ofP450d mutants were done as previously described [24,25,26,27,28].* All mutants were purified as a high spin form, and most of them were stable both for oxidized and reduced forms. It should be noted, that the Ly~"'~-Glu aLndyP3-G1u mutants were unstable at 37 "Cin termsof the Soret absorption spectrum of the CO-reduced form. L y ~ ' ' ~ - G l u , L y ~ ~ ~ ' - G l u , LAyr~g4~@~ ~-G-lGu,al nud, L y ~ ~ ~ ~ - G l u
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