Electron transfer between the FMN and heme domains of cytochrome P450BM-3 - Effects of substrate and CO

Cytochrome P450BM-3 has the P450 heme domain and FAD/FMN reductase domain linked together in a single polypeptide chain arranged as heme-FMN-FAD. In the accompanying article (Govindaraj, S,, and Poulos, T, L. (1997) J, Biol, Chem, 272, 7915-7921, we have described the preparation and characterization of the various domains of cytochrome P450BM-3, One reason for undertaking this study was to provide simpler systems for studying intramolecular electron transfer reactions, In particular, the heme-FMN version of P450BM-3 that is missing the FAD domain should prove useful in studying the FMN-to-heme electron transfer reaction, This version of P450BM-3 has been designated truncated P450BM-3 or BM3(t). In this study we have used laser flash photolysis techniques to generate the reduced semiquinone of 5-deazariboflavin which in turn reduces the FMN of BM3, to the semiquinone, FMN(radical anion), at a rate constant of 6600 s(-1), whereas the heme is not reduced by the 5-deazariboflavin radical, The reduction of the heme by FMN(radical anion) does not proceed in the absence of carbon monoxide (GO), whereas in the presence of CO the FMN(radical anion) to heme electron transfer rate constant is 18 s(-1). If a fatty acid substrate is present, this rate constant increases to 250 s(-1). Somewhat surprisingly, the rate of heme reduction also is dependent on [GO] which indicates that CO causes some change within the heme pocket and/or interaction between the heme and FMN domains that is required for intramolecular electron transfer.