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 BM3t. 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 BM3t to the semiquinone, FMN-, 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- does not proceed in the absence of carbon monoxide (CO), whereas in the presence of CO the FMN- 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 [CO] 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.