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. Nuclear magnetic resonance studies support the presence of a strong hydrogen bond between the backbone amide of Asn537 and FMN N5, the anionic ionization state of the hydroquinone, and for a change in the hybridization state of the N5 upon reduction. Replacement of Tyr536, which flanks the flavin ring, with a basic residue (histidine or arginine) did not significantly influence the redox properties of the FMN or the accumulation of the anionic semiquinone. The central residues of the type I' turn (Asn-Gly) were replaced with various combinations of glycine and alanine as a means of altering the turn and its interactions. Gly538 was found to be crucial in maintaining the type I' turn conformation of the loop and the strong H-bonding interaction at N5. The functional role of the tandem Pro-Pro sequence which anchors and possible "rigidifies" the loop was investigated through alanine replacements. Despite changes in the stabilities of the oxidized and hydroquinone redox states of the FMN, none of the replacements studied significantly altered the two-electron midpoint potentials. Pro541 does contribute to some degree to the strength of the N5 interaction and the formation of the anionic semiquinone. Unlike that of the flavodoxin, it would appear that the conformation of the FMN rather than the loop changes in response to reduction in this flavoprotein.

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.

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. In contrast, hydrogen bonding with the C(2)=O is one of the strongest among the flavoproteins investigated thus far. According to the crystal structure, the side-chain hydroxyl group of alphaSer254 serves as a hydrogen bond donor to the N(5) atom in the oxidized flavin cofactor in wETF. The replacement of alphaSer254 by cysteine by site-directed mutagenesis resulted in shifts in N(5)-relevant Raman bands in both the oxidized and anionic semiquinone states of the protein. These results confirm the presence of the hydrogen-bonding interaction at N(5) that is evident in the crystal structure of the oxidized protein and that it persists in the one-electron reduced state. The data suggest that these bands can serve as useful Raman markers for the N(5) interactions in both oxidation states of flavoproteins. The wETF displays unusually low frequencies of flavin ring I (o-xylene ring) relevant bands, which suggests a ring I microenvironment different from most of the other flavoproteins. As indicated by Raman data, the alphaS254C mutation changed the environment of ring I, perhaps as the consequence of changes in the mobility of the FAD domain of wETF. These unusual flavin-protein interactions may be associated with the unique redox properties of wETF.

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. The strength of this hydrogen bond was systematically altered by the substitution of alphaSer254 with threonine, cysteine, or alanine by site-directed mutagenesis. The anionic semiquinone form of the flavin, which is highly stabilized both thermodynamically and kinetically in the wild-type protein, was observed to accumulate in all three mutants. However, the midpoint potential for the first couple (Eox/sq) was significantly decreased for all of the mutants, and the kinetic barrier toward the reduction of the anionic semiquinone that is observed in the wild-type wETF was effectively abolished in the alphaS254T and alphaS254C mutants. Based on the observed changes in the Kd values and associated binding energies for the flavin, the amino acid replacements destabilize both the oxidized and semiquinone states of the flavin, but to a much greater extent for the anionic semiquinone state. The Eox/sq values for the alphaSer254 mutants follow a general trend with the strength of N(5) H-bond in the oxidized state as indicated by Raman spectral analyses. These results support the conclusion that the H-bonding interaction at the N(5) plays a key role in establishing the high Eox/sq and the unusually high stability of the anionic semiquinone state in wETF.

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., Perales-Alcon, A., Sancho, J., and Romero, A. (1996) Nat. Struct. Biol. 3, 329-332; Steensma, E., and van Mierlo, C. P. M. (1998) J. Mol. Biol. 282, 653-666]. Binding kinetics using stopped-flow spectrofluorimetry and phosphate competition studies were used to develop a model for flavin binding to the flavodoxin from Desulfovibrio vulgaris. In the presence of phosphate, the time course of fluorescence quenching associated with FMN binding to apoflavodoxin was biphasic, whereas riboflavin, which lacks the 5'-phosphate group of FMN, displayed monophasic binding kinetics. When the concentration of phosphate in solution was increased, the FMN binding rates of the two phases behaved differently; the rate of one phase decreased, while the rate of the other increased. A similar increase in the single phase associated with riboflavin binding was also observed. This has led to the following model. The binding of the flavin isoalloxazine ring to its subsite is dependent on the presence of a phosphate group in the phosphate-binding subsite. When phosphate is in the buffer solution, FMN can bind in either of two ways: by the initial insertion of the 5'-phosphate group followed by ring binding or, when inorganic phosphate from solution is bound, the insertion of the isoalloxazine ring first. Riboflavin, which lacks the phosphate moiety of FMN, binds only in the presence of inorganic phosphate, presumably due to the binding of this group in the phosphate-binding subsite. These results suggest that cooperative interactions exist between the phosphate subsite and the ring-binding region in the D. vulgaris flavodoxin that are necessary for isoalloxazine ring binding.

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. P. (2003) Biochemistry 42, 2307-2316]. In this model, the binding of the flavin isoalloxazine ring is dependent on the presence of a phosphate moiety in the phosphate-binding subsite, suggesting a cooperative interaction between that region and the ring-binding subsite. In the absence of inorganic phosphate, FMN can bind through the initial association of its 5'-phosphate group in the phosphate-binding subsite followed by insertion of the flavin ring. Because riboflavin lacks the 5'-phosphate group, it is unable to bind to this apoprotein in the absence of inorganic phosphate in solution. However, inorganic phosphate can enhance the rate of ring binding by occupying the phosphate-binding subsite. In this paper, NMR, near-UV circular dichroism (CD), and fluorescence spectroscopy provide evidence for a phosphate-induced conformational change within the isoalloxazine ring-binding subsite. Phosphate-dependent changes in the chemical shifts of 22 amide groups were observed in (1)H-(15)N HSQC NMR spectra. The majority of these groups are proximal to the phosphate-binding subsite or the loops that constitute the isoalloxazine ring-binding site. Also, a phosphate-dependent change in the environment or position of the Trp60 side chain was apparent in the NMR data and was confirmed by associated changes in the near-UV CD and tryptophan fluorescence spectra when compared to the spectra of the W60A mutant. These data suggest that phosphate, either the 5'-phosphate of the FMN or inorganic phosphate from solution, facilitates the movement of the side chain of Trp60 out of the isoalloxazine ring-binding site and other associated conformational changes, creating a population of apoflavodoxin that is capable of binding the isoalloxazine ring. This conformational switch may explain why some apoflavodoxins cannot bind riboflavin and also supports the "aromatic gate" model proposed from the crystal structure of the Anabaena apoflavodoxin [Genzor, C. G., Perales-Alcon, A., Sancho, J., and Romero, A. (1996) Nat. Struct. Biol. 3, 329-332].