The FMN-Binding Domain

In addition to these two key residues, a third aromatic side chain, PhelSl, lies close to the FMN 

while their catalytic efficiency with NADH was increased at least 500-fold. 

Three residues Ser457, Asp675, and Cys630, lie in close proximity to the FAD isoalloxazine ring in CPR, and have been closely examined 

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Shen, A.L., T.D. Porter, T.E. Wilson, and C.B. Kasper (1989). Structural analysis of the FMN binding domain of NADPH-cytochrome P-450 oxidore-ductase by site-directed mutagenesis. J. Biol. Chem. 264, 7584-7589. 

Zhao Q, Modi S, Smith G, Paine M, McDonagh PD, Wolf CR, Tew D, Lian LY, Roberts GC, Driessen HP (1999) Crystal structure of the FMN-binding domain of human cytochrome P450 reductase at 1.93 A resolution. Protein Sci 8 298-306... 

Given that the general structure of the FMN-binding domain of eukaiyotic CPR enzymes is highly related to that of microbial flavodoxins, it is perhaps not surprising that bacterial flavodoxins have been shown to act as redox... 

HainesDC, Sevrioukova IF, Peterson JA (2000) The FMN-binding domain of cytochrome P450BM-3 resolution, reconstitution, and flavin analogue substitution. Biochemistry 39 9419-9429... 

Fig. 3. A hypothetical ribozyme that can catalyze electron transfer. Aptamers than can bind NAD+ (and, hence, NADH) are selected, and the binding domain is mapped. An oligonucleotide tail that can bind to an unpaired region near the NAD-binding domain is attached to FMN. The bound FMN-oligonucleotide will be adjacent to NADH when it is bound in the active site of the ribozyme. Electron transfer should occur owing to the proximity of the two substrates. The rate of the reaction can be controlled by varying the length of the oligonucleotide tail to vary the distance between NADH and FMN substrate. Although this catalyst is extremely simple (and employs the same principles of catalysis found in nonenzymatic template-directed ligation reactions), it would nevertheless demonstrate the ability of RNA to catalyze reactions other than phosphodiester bond transfers.

Flavocytochrome b2 from Saccharomyces cerevisiae, a member of the FMN-dependent oxidoreductase superfamily, catalyzes the two-electron oxidation of lactate to pyruvate with subsequent electron-transfer to cytochrome c via the bound flavin [55], What distinguishes the enzyme from other family members is the N-terminal fusion of a heme-binding domain to the ySa-barrel structure, which hosts the primary active site. Rather than dumping the electrons from the reduced flavin hydroquinone onto molecular oxygen, they are transferred intramolecularly to the heme-binding domain and from there in a second intermolecular step to cytochrome c. 

The differences in quaternary structure as opposed to the preservation of tertiary structure of the NAD+ binding domains suggest that subunit associations are of more recent origin (9). Buehner et al. 17) used the preservation of the Q axis in GAPDH, LDH, and s-MDH, and the close resemblance of the dimer of s-MDH to one-half of the LDH molecule, to suggest an evolutionary tree (Fig. 12). Also included in Fig. 12 is the relationship of an FMN mononucleotide binding unit in flavodoxin 24) to the dehydrogenases. 

The nucleotides NAD, NADH, FAD, and FMN are all known to be inhibitors. The structure of this enzyme was solved (74) and was found to have two repeating structural domains along a (probably) single polypeptide chain. Each domain contained a fold reminiscent of the nucleotide binding domains in the dehydrogenases. It remains to be seen whether nucleotides bind in a manner suggested by the structural similarity. 

Figure 34 Stereoview of the residues defining the ammonia tunnei in Fd-dependent Synechocystis GitS (1OFE) and crystaiiographic waters (red spheres). Residue side chains are shown as sticks. Coioring C - magenta (centrai domain residues) and green (FMN-binding domain residues), 0-red, N -biue, P-orange, and S-yeiiow. image rendered in PYMOL.

Addition of cytochrome hi core to a solution of TNS does not modify the fluorescence of the probe. However, upon addition of the cytochrome to a solution of FDH-TNS complex, an increase in TNS fluorescence was observed, as the result of an increase in the affinity between the probe and the flavoprotein (A.ex, 320 nm) (Albani, 1993). X-ray diffraction studies have indicated that FMN is buried in the flavin-binding domain of flavocytochrome hi (Zia et al. 1990), and fluorescence studies have shown that binding of TNS to the FDH does not induce any release of the flavin from its binding site (Albani, 1993). Titration of a constant concentration of FDH-TNS complex with cytochrome hi core yields a sigmoidal curve for the TNS intensity increase (Fig. 4.18) (Albani, 1997). Thus, interaction between cytochrome hi core and FDH is cooperative. 

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