Effect of the Insertion of a Glycine Residue into the Loop Spanning Residues 536-541 on the Semiquinone State and Redox Properties of the Flavin Mononucleotide-Binding Domain of Flavocytochrome P450BM-3 from Bacillus megaterium.

Despite sharing sequence and structural similarities with other diflavin reductases such as NADPH-cytochrome P450 reductase (CPR) and nitric oxide synthase, flavocytochrome P450BM-3 displays some unique redox and electron transferring properties, including the inability to thermodynamically stabilize the neutral semiquinone (SQ) state of the flavin mononucleotide (FMN) cofactor. Rather, the anionic SQ species is only transiently formed during rapid reduction. Why is this? The absence of a conserved glycine residue and, as a consequence, the shorter and less flexible cofactor-binding loop in P450BM-3 represents a notable difference from other diflavin reductases and the structurally related flavodoxin. This difference may facilitate the formation of a strong hydrogen bond between backbone amide NH group of Asn537 and N5 of the oxidized FMN, an interaction not found in the other proteins. In the flavodoxin, the conserved glycine residue plays a crucial role in a redox-linked conformational change that contributes to the thermodynamic stabilization of the neutral SQ species of the FMN through the formation of a hydrogen bond with the N5H group of the flavin. In this study, a glycine residue was inserted after Tyr536 in the loop within the isolated FMN-binding domain as well as the diflavin reductase domain of P450BM-3, a position equivalent to Gly141 in human CPR. As a result, the insertion variant was observed to accumulate the neutral form of the FMN SQ species much like CPR. The midpoint potential for the SQ/HQ couple decreased by 68 mV, while that for the OX/SQ couple remained unchanged. (15)N NMR data provide evidence of the disruption of the hydrogen bond between the backbone amide group of Asn537 and the N5 atom in the oxidized state of the FMN. Molecular models suggest that the neutral FMN SQ could be stabilized through hydrogen bonding with the backbone carbonyl group of the inserted glycine residue in a manner similar to that of CPR and the flavodoxin. The insertion of the glycine at the same location within the diflavin domain resulted in a purified protein that retained nearly stoichiometric levels of bound FAD but tended to lose the FMN cofactor. This preparation retained one-third of the ferricyanide reductase activity but <1% of the cytochrome c reductase activity of the wild type. However, the insertion variant reconstituted with FMN regained nearly half of the wild-type cytochrome c reductase activity. These results demonstrate the importance of the unique structural characteristics of the shorter loop in P450BM-3 in establishing the unique redox properties of the FMN in this protein but not its general cytochrome reductase activity.