Abstract
Transfer of reducing equivalents from NADPH to the cytochromes P450 is mediated by NADPH-cytochrome P450 oxidoreductase, which contains stoichiometric amounts of tightly bound FMN and FAD. Hydrogen bonding and van der Waals interactions between FAD and amino acid residues in the FAD binding site of the reductase serve to regulate both flavin binding and reactivity. The precise orientation of key residues (Arg(454), Tyr(456), Cys(472), Gly(488), Thr(491), and Trp(677)) has been defined by x-ray crystallography (Wang, M., Roberts, D. L., Paschke, R., Shea, T. M., Masters, B. S., Kim, J.-J. P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8411-8416). The current study examines the relative contributions of these residues to FAD binding and catalysis by site-directed mutagenesis and kinetic analysis. Mutation of either Tyr(456), which makes van der Waals contact with the FAD isoalloxazine ring and also hydrogen-bonds to the ribityl 4'-hydroxyl, or Arg(454), which bonds to the FAD pyrophosphate, decreases the affinity for FAD 8000- and 25,000-fold, respectively, with corresponding decreases in cytochrome c reductase activity. In contrast, substitution of Thr(491), which also interacts with the pyrophosphate grouping, had a relatively modest effect on both FAD binding (100-fold decrease) and catalytic activity (2-fold decrease), while the G488L mutant exhibited, respectively, 800- and 50-fold decreases in FAD binding and catalytic activity. Enzymic activity of each of these mutants could be restored by addition of FAD. Kinetic properties and the FMN content of these mutants were not affected by these substitutions, with the exception of a 3-fold increase in Y456S K(m)(cyt )(c) and a 70% decrease in R454E FMN content, suggesting that the FMN- and FAD-binding domains are largely, but not completely, independent. Even though Trp(677) is stacked against the re-face of FAD, suggesting an important role in FAD binding, deletion of both Trp(677) and the carboxyl-terminal Ser(678) decreased catalytic activity 50-fold without affecting FAD content.
Highlights
The flavoprotein NADPH-cytochrome P450 oxidoreductase, which mediates the transfer of electrons from NADPH to the cytochromes P450 and other microsomal electron acceptors, is one of a family of FMN- and FAD-containing enzymes (1– 4) that includes the bacterial sulfite reductase ␣-subunit and P450-BM3 (5, 6) as well as the mammalian enzymes nitricoxide synthase and methionine synthase reductase (7, 8)
The current study examines the relative contributions of these residues to FAD binding and catalysis by sitedirected mutagenesis and kinetic analysis
Mutation of either Tyr[456], which makes van der Waals contact with the FAD isoalloxazine ring and hydrogen-bonds to the ribityl 4-hydroxyl, or Arg[454], which bonds to the FAD pyrophosphate, decreases the affinity for FAD 8000- and 25,000-fold, respectively, with corresponding decreases in cytochrome c reductase activity
Summary
The flavoprotein NADPH-cytochrome P450 oxidoreductase, which mediates the transfer of electrons from NADPH to the cytochromes P450 and other microsomal electron acceptors, is one of a family of FMN- and FAD-containing enzymes (1– 4) that includes the bacterial sulfite reductase ␣-subunit and P450-BM3 (5, 6) as well as the mammalian enzymes nitricoxide synthase and methionine synthase reductase (7, 8). X-ray crystallographic studies confirmed this hypothesis, demonstrating that this interconnecting region is organized into a unique structural domain that interacts with both flavin binding regions and appears to be responsible for aligning the isoalloxazine rings end to end with the dimethylbenzene rings facing each other at a spacing of approximately 4 Å (15). The isoalloxazine ring of FAD is sandwiched between two aromatic groups, with Trp[677] stacked against the re-face and Tyr[456] positioned at a 60° angle to the si-face, an arrangement similar to that of the FMN binding site (15). Main chain atoms of Ser[457], Tyr[455], Cys[472], and Val[474] are within hydrogen bonding distance of the isoalloxazine ring. Unlike FMN, FAD binding does not require the presence of an aromatic group stacked against the re-face of the isoalloxazine ring
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