FAD-binding site of glutathione reductase

The FAD-binding site in dimeric glutathione reductase has been elucidated by application of sequence and X-ray analyses in parallel. The geometry was derived from a multiple isomorphous replacement map of 2 Å resolution. FAD binds in a rather elongated conformation with the flavin portion in the centre of one subunit and the adenine portion extending to its surface. The FMN moiety of FAD is completely buried in the protein, whereas the AMP moiety is partly accessible from the solvent. There are three strong dipoles in the vicinity of flavin, Lys66:Glu201 at N-5, Arg291:W-3:Asp331 close to C-8α, and His467: Glu472 near C-2, with the positive partners always pointing to the flavin moiety. The substrate, NADPH, releases its reduction equivalents to the re-face of flavin. Flavin passes them on to the redoxactive disulphide bridge Cys58: Cys63 at its si-face. This bridge has an unusual conformation. The negative charges of the pyrophosphate portion of FAD are not compensated by positively charged sidechains of the protein. On the contrary, there are two carboxylates nearby; Glu50 binding to the ribose, and Asp331 contacting the ribitol and one phosphate. Since electroneutrality is required, it is likely that at least one of the observed globules of electron density at the pyrophosphate is a cation. Adenine binds in a shallow nonpolar pocket with N-1A. N-3A, N-6αA forming hydrogen bonds to the protein. The conformation of FAD is compared with those of other protein-bound nucleotides. Sequence similarities with three other FAD-binding proteins are discussed.