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 P450 BM-3 was accomplished using trichloroacetic acid. The apoprotein thus prepa red was shown to rebind FMN to regenerate the original holoprotein as indic ated by both spectroscopy and activity measurements. To better understand h ow the protein/flavin interaction might contribute to reactivity, the assoc iation process was studied in detail. Fluorescence quenching was used to me asure a dissociation constant of the flavin-protein complex of 31 nM, compa rable to FMN-containing proteins of similar reactivity and higher than that of flavodoxins. Stopped-flow kinetics were performed, and a multistep bind ing 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 analogu es for the native FMN cofactor using 8-chloro-FMN and 8-amino-FMN. Both wer e found to bind efficiently to the protein with only minor variations in af finity. Reductive titrations established that, as in the native FMN-contain ing FMN-binding domain, the 8-amino-FMN-substituted domain does not produce a stable one-electron-reduced species during titration with sodium dithion ite. The 8-chloro-FMN-substituted domain, however, had sufficiently altered redox properties to form a stable red anionic semiquinone. The 8-chloro-FM N-substituted FMN-binding domain was shown in reconstituted systems to reta in most of the cytochrome c reductase activity of the native domain but onl y a very small amount of palmitic acid hydroxylase activity. The 8-amino-FM N-substituted FMN-binding domain showed no palmitic acid hydroxylase activi ty and only 30% of the native cytochrome c reductase activity, demonstratin g the importance of thermodynamics to the mechanism of this protein.