Analysis of the structure of the flavin binding sites of flavocytochrome P450BM3 using surface enhanced resonance Raman scattering

Flavocytochrome P450 BM3, an FMN-deficient mutant (G570D), the component re ductase and an FAD-containing domain were studied using surface enhanced re sonance Raman scattering (SERRS). They were compared to spectra obtained fr om the free flavins FAD and FMN. For the holoenzyme and reductase domain, F MN is displaced during SERRS analysis. However, studies with the G570D muta nt indicate that FAD is retained in its active site. Analysis of SERRS freq uencies and intensities provides information on the nature of the flavin bi nding site and the planarity of the ring, and enables an interpretation of the hydrogen bonding environment around ring III of the isoalloxazine moiet y. Hydrogen bonding is strong at N-3-H, C-2=O and C-4=O, but weak at N-5. S tructural alteration of the FAD domain of P450 BM 3 is caused by removal of the FMN-binding domain. Further, the hydrogen bond at N-3-H is lost and th at at C-2=O is weakened and the isoalloxazine ring system in the FAD domain appears to adopt a more planar arrangement. Alterations in the environment of the FAD in its isolated domain are likely to relate to changes in the r edox properties and suggest a close structural interplay of FAD with the FM N-binding domain in intact flavocytochrome P450 BM3.