Structural prototypes for an extended family of flavoprotein reductases: Comparison of phthalate dioxygenase reductase with ferredoxin reductase and ferredoxin

Abstract

The structure of phthalate dioxygenase reductase (PDR), a monomeric iron-sulfur flavoprotein that delivers electrons from NADH to phthalate dioxygenase, is compared to ferredoxin-NADP+ reductase (FNR) and ferredoxin, the proteins that reduce NADP+ in the final reaction of photosystem I. The folding patterns of the domains that bind flavin, NAD(P), and [2Fe-2S] are very similar in the two systems. Alignment of the X-ray structures of PDR and FNR substantiates the assignment of features that characterize a family of flavoprotein reductases whose members include cytochrome P-450 reductase, sulfite and nitrate reductases, and nitric oxide synthase. Hallmarks of this subfamily of flavoproteins, here termed the FNR family, are an antiparallel 0-barrel that binds the flavin prosthetic group, and a characteristic variant of the classic pyridine nucleotide-binding fold. Despite the similarities between FNR and PDR, attempts to model the structure of a dissociable FNR:ferredoxin complex by analogy with PDR reveal features that are at odds with chemical crosslinking studies (Zanetti, G., Morelli, D., Ronchi, S . , Negri, A., Aliverti, A., & Curti, B., 1988, Biochemistry 27, 3753-3759).Differences in the binding sites for flavin and pyridine nucleotides determine the nucleotide specificities of FNR and PDR. The specificity of FNR for NADP' arises primarily from substitutions in FNR that favor interactions with the 2' phosphate of NADP'. Variations in the conformation and sequences of the loop adjoining the flavin phosphate affect the selectivity for FAD versus FMN.The midpoint potentials for reduction of the flavin and [2Fe-2S] groups in PDR are higher than their counterparts in FNR and spinach ferredoxin, by about 120 mV and 260 mV, respectively. Comparisons of the structure of PDR with spinach FNR and with ferredoxin from Anabaena 7120, along with calculations of electrostatic potentials, suggest that local interactions, including hydrogen bonds, are the dominant contributors to these differences in potential.

Probing the NADPH-binding site of Escherichia coli flavodoxin oxidoreductase

“…For instance, in the bovine NADH-cytochrome b & reductase, an aspartate replaces the serine. This is also the case in corn nitrate reductase and other related NAD + -dependent systems [10,13]. By analogy with the NADPH-and NADH-dependent glutathione reductases [20], the aspartate side chains may be involved in hydrogen bonding to the 2h hydroxy group of the adenosine ribose in NADH, i.e.…”

“…The FLDR structure has also led to the suggestion that another cluster of arginine residues (R144, R174 and R184) is involved in the interaction with the pyridine dinucleotide cofactor (NADPH ; Figure 1) ; although the cofactor was not co-crystallized with the enzyme [9]. In FLDR crystals, citrate precipitant was located bound in a site proposed to accommodate the adenine ribose of NADPH (based on homology with the spinach FNR) [9,10], and was seen to interact with amino acid R174, a conserved residue described as part of a ' fingerprint ' region for NADP + -binding members of the FNRlike family of flavoenzymes [13].…”

Structure of the Mutant E92K of [2Fe–2S] Ferredoxin I from Spinacia oleracea at 1.7 Å Resolution

“…Homology-modelling studies based on the structure of phthalate dioxygenase reductase suggest that residue 92 is unlikely to be directly involved in the interactions between Fd and ferredoxin±NADP + reductase (Correll et al, 1993). On the other hand, the similarity between the structure of the E92K mutant and that of the other ferredoxins implies that the mutation does not induce substantial structural changes that could be responsible for a less favourable interaction of the mutant Fd I with the reductase.…”

Viscosity Effects on Eukaryotic Nitrate Reductase Activity

“…NaR is a member of the sulfite oxidase (SOX) structure family of Mo-MPT-containing enzymes with which it shares high sequence similarity for the Mo-containing module (5). The Cyt b reductase fragment (CbR) of NaR for which the structure has been determined (6, 7) is related to the ferredoxin NADP ϩ reductase structural family (8). The Cyt b of NaR is related to mammalian Cyt b 5 and SOX Cyt b with which it shares a high sequence homology (1,9).…”