The FMN-binding domain of cytochrome P450BM-3: resolution, reconstitution, and flavin analogue substitution.

Cytochrome P450BM-3 is a self-sufficient bacterial protein containing three naturally fused domains which bind either heme, FMN, or FAD. Resolution of protein and FMN from the isolated FMN-containing domain of cytochrome P450Betamicro-3 was accomplished using trichloroacetic acid. The apoprotein thus prepared was shown to rebind FMN to regenerate the original holoprotein as indicated by both spectroscopy and activity measurements. To better understand how the protein/flavin interaction might contribute to reactivity, the association process was studied in detail. Fluorescence quenching was used to measure a dissociation constant of the flavin-protein complex of 31 nM, comparable to FMN-containing proteins of similar reactivity and higher than that of flavodoxins. Stopped-flow kinetics were performed, and a multistep binding process was indicated, with an initial k(on) value of 1.72 x 10(5) M(-)(1) s(-)(1). Preparation of the apoprotein allowed substitution of flavin analogues for the native FMN cofactor using 8-chloro-FMN and 8-amino-FMN. Both were found to bind efficiently to the protein with only minor variations in affinity. Reductive titrations established that, as in the native FMN-containing FMN-binding domain, the 8-amino-FMN-substituted domain does not produce a stable one-electron-reduced species during titration with sodium dithionite. The 8-chloro-FMN-substituted domain, however, had sufficiently altered redox properties to form a stable red anionic semiquinone. The 8-chloro-FMN-substituted FMN-binding domain was shown in reconstituted systems to retain most of the cytochrome c reductase activity of the native domain but only a very small amount of palmitic acid hydroxylase activity. The 8-amino-FMN-substituted FMN-binding domain showed no palmitic acid hydroxylase activity and only 30% of the native cytochrome c reductase activity, demonstrating the importance of thermodynamics to the mechanism of this protein.