STRUCTURAL PROTOTYPES FOR AN EXTENDED FAMILY OF FLAVOPROTEIN REDUCTASES - COMPARISON OF PHTHALATE DIOXYGENASE REDUCTASE WITH FERREDOXIN REDUCTASE AND FERREDOXIN

The structure of phthalate dioxygenase reductase (PDR), a monomeric ir on-sulfur flavoprotein that delivers electrons from NADH to phthalate dioxygenase, is compared to ferredoxin-NADP+ reductase (FNR) and ferre doxin, the proteins that reduce NADP+ in the final reaction of photosy stem I. The folding patterns of the domains that bind flavin, NAD(P), and 12Fe-2S] are very similar in the two systems. Alignment of the X-r ay structures of PDR and FNR substantiates the assignment of features that characterize a family of flavoprotein reductases whose members in clude cytochrome P-450 reductase, sulfite and nitrate reductases, and nitric oxide synthase. Hallmarks of this subfamily of flavoproteins, h ere termed the FNR family, are an antiparallel beta-barrel that binds the flavin prosthetic group, and a characteristic variant of the class ic pyridine nucleotide-binding fold. Despite the similarities between FNR and PDR, attempts to model the structure of a dissociable FNR:ferr edoxin complex by analogy with PDR reveal features that are at odds wi th chemical crosslinking studies (Zanetti, G., Morelli, D., Ronchi, S. , Negri, A., Aliverti, A., & Curti, B., 1988, Biochemistry 27, 3753-37 59). Differences in the binding sites for flavin and pyridine nucleoti des determine the nucleotide specificities of FNR and PDR. The specifi city of FNR for NADP+ arises primarily from substitutions in FNR that favor interactions with the 2' phosphate of NADP+. Variations in the c onformation and sequences of the loop adjoining the flavin phosphate a ffect the selectivity for FAD versus FMN. The midpoint potentials for reduction of the flavin and [2Fe-2S] groups in PDR are higher than the ir counterparts in FNR and spinach ferredoxin, by about 120 mV and 260 mV, respectively. Comparisons of the structure of PDR with spinach FN R and with ferredoxin from Anabaena 7120, along with calculations of e lectrostatic potentials, suggest that local interactions, including hy drogen bonds, are the dominant contributors to these differences in po tential.