Functional characterization of the re-face loop spanning residues 536-541 and its interactions with the cofactor in the flavin mononucleotide-binding domain of flavocytochrome P450 from Bacillus megaterium.

Flavocytochrome P450BM-3, a bacterial monooxygenase, contains a flavin mononucleotide-binding domain bearing a strong structural homology to the bacterial flavodoxin. The flavin mononucleotide (FMN) serves as the one-electron donor to the heme iron, but in contrast to the electron transfer mechanism of mammalian cytochrome P450 reductase, the FMN semiquinone state is not thermodynamically stable and appears transiently as the anionic rather than the neutral form. A unique loop region comprised of residues (536)Y-N-G-H-P-P(541), which forms a type I' reverse turn and provides several interactions with the FMN isoalloxazine ring, was targeted in this study.

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.

Modulation of the redox properties of the flavin cofactor through hydrogen-bonding interactions with the N(5) atom: role of alphaSer254 in the electron-transfer flavoprotein from the methylotrophic bacterium W3A1.

The functional effects of hydrogen-bonding interactions at the N(5) atom of the flavin cofactors in the oxidized state have not been well established in flavoproteins. The unique properties of the electron-transfer flavoprotein from the methylotrophic bacteria W3A1 (wETF) were used to advantage in this study to evaluate this interaction. In wETF, the side-chain hydroxyl group of alphaSer254 serves as a hydrogen bond donor to the N(5) atom in the oxidized state of the flavin.

Nonresonance Raman study of the flavin cofactor and its interactions in the methylotrophic bacterium W3A1 electron-transfer flavoprotein.

Nonresonance Raman spectroscopy has been used to investigate the protein-flavin interactions of the oxidized and anionic semiquinone states of the electron-transfer flavoprotein from the methylotrophic bacteria W3A1 (wETF) in solution. Several unique features of oxidized wETF were revealed from the Raman data. The unusually high frequency of the Raman band for the C(4)=O of the flavin suggests that hydrogen-bonding interactions with the C(4)O are very weak or nonexistent in wETF.

Mechanism of flavin mononucleotide cofactor binding to the Desulfovibrio vulgaris flavodoxin. 2. Evidence for cooperative conformational changes involving tryptophan 60 in the interaction between the phosphate- and ring-binding subsites.

A mechanism has been proposed for the binding of flavin mononucleotide (FMN) and riboflavin to the apoflavodoxin from Desulfovibrio vulgaris [Murray, T. A., and Swenson, R.

Mechanism of flavin mononucleotide cofactor binding to the Desulfovibrio vulgaris flavodoxin. 1. Kinetic evidence for cooperative effects associated with the binding of inorganic phosphate and the 5'-phosphate moiety of the cofactor.

The pathway(s) by which the flavin cofactor binds to the apoflavoprotein is the subject of some debate. The crystal and NMR structures of several different flavodoxins have provided some insight, although there is disagreement about the location of the initial interaction between the flavin mononucleotide (FMN) and the apoflavodoxin and the degree of protein conformational change associated with cofactor binding [Genzor, C. G.