Phthalate Dioxygenase Reductase Research Articles

Characterization of phthalate reductase from Ralstonia eutropha CH34 and in silico study of phthalate dioxygenase and phthalate reductase interaction

Microbial degradation is a natural means to combat ubiquitous, harmful and non-covalently bound plasticizers, phthalic acid esters (PAEs), due to their slower photo-degradation and hydrolysis rates. In microbial aerobic degradation of phthalates, phthalate dioxygenase system (PDS) belongs to a large family of Rieske non-heme iron oxygenases with type1A electron-transfer system. Two important proteins of this system include: phthalate dioxygenase reductase (PDR), a flavo-iron-sulfur protein having NADH-dependent oxidoreductase activity; and phthalate dioxygenase oxygenase (PDO), a non-heme iron protein with oxygenase activity. In the present study, phthalate dioxygenase reductase (RePDR) from Ralstonia eutropha CH34, has been cloned, expressed and purified. Further, in order to understand the interactions between RePDO and RePDR, protein-protein docking has been carried out. The homology model structures of the individual proteins served as an input for protein-protein interaction studies and helps in the prediction of complex structure. Important interface residues from both the individual counterparts, i.e. RePDO and RePDR have been identified and the evolutionary relationship between these important interface residues has also been established. Moreover, the stability of these interactions involving highly conserved interface residues, highlights their involvement in structurally conserved interactions and play significant role in phthalate degrading system.

Mechanistic basis of electron transfer to cytochromes p450 by natural redox partners and artificial donor constructs.

Cytochromes P450 (P450s) are hemoproteins catalyzing oxidative biotransformation of a vast array of natural and xenobiotic compounds. Reducing equivalents required for dioxygen cleavage and substrate hydroxylation originate from different redox partners including diflavin reductases, flavodoxins, ferredoxins and phthalate dioxygenase reductase (PDR)-type proteins. Accordingly, circumstantial analysis of structural and physicochemical features governing donor-acceptor recognition and electron transfer poses an intriguing challenge. Thus, conformational flexibility reflected by togging between closed and open states of solvent exposed patches on the redox components was shown to be instrumental to steered electron transmission. Here, the membrane-interactive tails of the P450 enzymes and donor proteins were recognized to be crucial to proper orientation toward each other of surface sites on the redox modules steering functional coupling. Also, mobile electron shuttling may come into play. While charge-pairing mechanisms are of primary importance in attraction and complexation of the redox partners, hydrophobic and van der Waals cohesion forces play a minor role in docking events. Due to catalytic plasticity of P450 enzymes, there is considerable promise in biotechnological applications. Here, deeper insight into the mechanistic basis of the redox machinery will permit optimization of redox processes via directed evolution and DNA shuffling. Thus, creation of hybrid systems by fusion of the modified heme domain of P450s with proteinaceous electron carriers helps obviate the tedious reconstitution procedure and induces novel activities. Also, P450-based amperometric biosensors may open new vistas in pharmaceutical and clinical implementation and environmental monitoring.

Stress response of Burkholderia cepacia WZ1 exposed to quinclorac and the biodegradation of quinclorac

Quinclorac is an effective herbicide, but its residue is known to be a major environmental pollutant. Therefore, isolating quinclorac-degrading bacteria, exploring the mechanisms of degradation and analyzing the proteins introduced by the pollutant, would have scientific value and practical importance. We used quinclorac-degrading bacteria WZ1 (an opportunistic pathogen with broad substrate characteristic) as the test strain, and used several techniques such as two-dimensional electrophoresis, gas chromatography–mass spectrometry and Northern Blotting to study the stress response of the strain upon environmental stress and the biodegradation of quinclorac. The results showed that strain WZ1 secreted some protective proteins such as Mn-SOD, Heat-shock protein and Taurine dioxygenase to deal with the stress of quinclorac; while the expression of proteins DsbA and Gmet_1484 related to bacterial pathogenesis decreased significantly when exposed to quinclorac (which could be used to indicate that bacterial pathogenicity was attenuated). At the same time, quinclorac also induced the expression of phthalate dioxygenase reductase and chlorocatechol 1, 2-dioxygenase. They are the key proteins participating in the degradation of quinclorac, which was proved by the Northern Blotting. With the information gathered, we attempted to define the degradation pathway of quinclorac.

High levels of expression of the iron–sulfur proteins phthalate dioxygenase and phthalate dioxygenase reductase in Escherichia coli

Phthalate dioxygenase (PDO), a hexamer with one Rieske-type [2Fe–2S] and one Fe (II)—mononuclear center per monomer, and its reductase (PDR), which contains flavin mononucleotide and a plant-type ferredoxin [2Fe–2S] center, are expressed by Burkholderia cepacia at ∼30 mg of crude PDO and ∼1 mg of crude PDR per liter of cell culture when grown with phthalate as the main carbon source. A high level expression system in Escherichia coli was developed for PDO and PDR. Optimization relative to E. coli cell line, growth parameters, time of induction, media composition, and iron–sulfur additives resulted in yields of about 1 g/L for PDO and about 0.2 g/L for PDR. Protein expression was correlated to the increase in pH of the cell culture and exhibited a pronounced (variable from 5 to 20 h) lag after the induction. The specific activity of purified PDO did not depend on the pH of the cell culture when harvested. However, when the pH of the culture reached 8.5–9, a large fraction of the PDR that was expressed lacked its ferredoxin domain, presumably because of proteolysis. Termination of growth while the pH of the cell culture was <8 decreased the fraction of proteolyzed enzyme, whereas yields of the unclipped PDR were only marginally lower. Overall, changes in pH of the cell culture were found to be an excellent indicator of the overall level of native protein expression. Its monitoring allowed the real time tracking of the protein expression and made it possible to tailor the expression times to achieve a combination of high quality and high yield of protein.

Rates of the phthalate dioxygenase reaction with oxygen are dramatically increased by interactions with phthalate and phthalate oxygenase reductase.

The phthalate dioxygenase system, which catalyzes the dihydroxylation of phthalate to form its cis-dihydrodiol (DHD), has two components: phthalate dioxygenase (PDO), a multimer with one Rieske-type [2Fe-2S] and one Fe(II) center per monomer, and phthalate dioxygenase reductase (PDR), which contains flavin mononucleotide (FMN) and a plant-like ferredoxin [2Fe-2S] center. PDR is responsible for transferring electrons from NADH to the Rieske center of PDO, and the Rieske center supplies electrons to the mononuclear center for the oxygenation of substrate. Reduced PDO (PDO(red)) that lacks Fe(II) at the mononuclear metal site (PDO-APO) reacts slowly with O(2) (1.4 x 10(-3) s(-1) at 125 microM O(2) and 22 degrees C), presumably in a direct reaction with the Rieske center. Binding of phthalate and/or PDR(ox) to reduced PDO-APO increases the reactivity of the Rieske center with O(2). When no PDR or phthalate is present, the oxidation of the Rieske center in native PDO(red) [which contains Fe(II) at the mononuclear site] occurs in two phases (approximately 1 and 0.1 s(-1) at 125 mM O(2), 23 degrees C), both much faster than in the absence of Fe(II), presumably because in this case O(2) reacts at the mononuclear Fe(II). Addition of PDR(ox) to native PDO(red) resulted in a large fraction of the Rieske center being oxidized at 5 s(-1), and the addition of phthalate resulted in about 70% of the reaction proceeding at 42 s(-1). With both PDR(ox) and phthalate present, most of the PDO(red) (approximately 80-85%) oxidizes at 42 s(-1), with the remaining oxidizing at approximately 5 s(-1). Thus, the binding of phthalate or PDR(ox) to PDO(red) each results in greater reactivity of PDO with O(2). The presence of both the substrate and PDR was synergistic, making PDO fully catalytically active. A model that explains the observed effects is presented and discussed in terms of PDO subunit cooperativity. It is proposed that, during oxidation of reduced PDO, each of two Rieske centers on separate subunits transfers an electron to the Fe(II) mononuclear center on a third subunit. This explanation is consistent with the observed multiphasic kinetics of the oxidation of the Rieske center and is being further tested by product analysis experiments.

X-ray Crystal Structure of Benzoate 1,2-Dioxygenase Reductase from Acinetobacter sp. Strain ADP1

One of the major processes for aerobic biodegradation of aromatic compounds is initiated by Rieske dioxygenases. Benzoate dioxygenase contains a reductase component, BenC, that is responsible for the two-electron transfer from NADH via FAD and an iron–sulfur cluster to the terminal oxygenase component. Here, we present the structure of BenC from Acinetobacter sp. strain ADP1 at 1.5 Å resolution. BenC contains three domains, each binding a redox cofactor: iron–sulfur, FAD and NADH, respectively. The [2Fe–2S] domain is similar to that of plant ferredoxins, and the FAD and NADH domains are similar to members of the ferredoxin:NADPH reductase superfamily. In phthalate dioxygenase reductase, the only other Rieske dioxygenase reductase for which a crystal structure is available, the ferredoxin-like and flavin binding domains are sequentially reversed compared to BenC. The BenC structure shows significant differences in the location of the ferredoxin domain relative to the other domains, compared to phthalate dioxygenase reductase and other known systems containing these three domains. In BenC, the ferredoxin domain interacts with both the flavin and NAD(P)H domains. The iron–sulfur center and the flavin are about 9 Å apart, which allows a fast electron transfer. The BenC structure is the first determined for a reductase from the class IB Rieske dioxygenases, whose reductases transfer electrons directly to their oxygenase components. Based on sequence similarities, a very similar structure was modeled for the class III naphthalene dioxygenase reductase, which transfers electrons to an intermediary ferredoxin, rather than the oxygenase component.

Crystal Structure of Riboflavin Synthase

Background: Riboflavin synthase catalyzes the dismutation of two molecules of 6,7-dimethyl-8-(1′-D-ribityl)-lumazine to yield riboflavin and 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine. The homotrimer of 23 kDa subunits has no cofactor requirements for catalysis. The enzyme is nonexistent in humans and is an attractive target for antimicrobial agents of organisms whose pathogenicity depends on their ability to biosynthesize riboflavin. Results: The first three-dimensional structure of the enzyme was determined at 2.0 Å resolution using the multiwavelength anomalous diffraction (MAD) method on the Escherichia coli protein containing selenomethionine residues. The homotrimer consists of an asymmetric assembly of monomers, each of which comprises two similar β barrels and a C-terminal α helix. The similar β barrels within the monomer confirm a prediction of pseudo two-fold symmetry that is inferred from the sequence similarity between the two halves of the protein. The β barrels closely resemble folds found in phthalate dioxygenase reductase and other flavoproteins. Conclusions: The three active sites of the trimer are proposed to lie between pairs of monomers in which residues conserved among species reside, including two Asp-His-Ser triads and dyads of Cys-Ser and His-Thr. The proposed active sites are located where FMN (an analog of riboflavin) is modeled from an overlay of the β barrels of phthalate dioxygenase reductase and riboflavin synthase. In the trimer, one active site is formed, and the other two active sites are wide open and exposed to solvent. The nature of the trimer configuration suggests that only one active site can be formed and be catalytically competent at a time.

Novel organization of the genes for phthalate degradation from Burkholderia cepacia DBO1.

Burkholderia cepacia DBO1 is able to utilize phthalate as the sole source of carbon and energy for growth. Two overlapping cosmid clones containing the genes for phthalate degradation were isolated from this strain. Subcloning and activity analysis localized the genes for phthalate degradation to two separate regions on the cosmid clones. Analysis of the nucleotide sequence of these two regions showed that the genes for phthalate degradation are arranged in at least three transcriptional units. The gene for phthalate dioxygenase reductase (ophA1) is present by itself, while the genes for an inactive transporter (ophD) and 4,5-dihydroxyphthalate decarboxylase (ophC) are linked and the genes for phthalate dioxygenase oxygenase (ophA2) and cis-phthalate dihydrodiol dehydrogenase (ophB) are linked. ophA1 and ophDC are adjacent to each other but are transcribed in opposite directions, while ophA2B is located 4 kb away. The genes for the oxygenase and reductase components of phthalate dioxygenase are located approximately 7 kb away from each other. The gene for the putative phthalate permease contains a frameshift mutation in contrast to genes for other permeases. Strains deleted for ophD are able to transport phthalate into the cell at rates equivalent to that of the wild-type organism, showing that this gene is not required for growth on phthalate.

Incorporating Protein Environments in Density Functional Theory: A Self-Consistent Reaction Field Calculation of Redox Potentials of [2Fe2S] Clusters in Ferredoxin and Phthalate Dioxygenase Reductase

An approach to calculating molecular electronic structures of active-site clusters in the presence of protein environments has been developed. The active-site cluster is treated by density functional theory. The protein field, together with the reaction field arising mainly from solvent, is obtained from a finite-difference solution to the Poisson−Boltzmann equation with three dielectric regions, and then these are coupled to the density functional calculation by a self-consistent iterative procedure. The method is applied to compute redox potentials of ferredoxin from Anabaena 7120 and phthalate dioxygenase reductase (PDR) from Pseudomonas cepacia, both having similar [Fe2S2(SR)4] active-site clusters. The calculated redox potentials, −1.007 V and −0.812 V in 0.05 M ionic strength for ferredoxin and PDR, respectively, deviate significantly from experimental values of −0.440 and −0.174 V. However, the calculated data reproduce the experimental trend fairly well. The calculated redox potential for PDR is 195 mV more positive than that for ferredoxin, comparing very well with the experimental value of 266 mV. The energy decomposition scheme reveals that the protein field plays a key role in differentiating the redox potentials of these two proteins.

The three-dimensional structure of flavodoxin reductase from Escherichia coli at 1.7 å resolution

Flavodoxin reductase from Escherichia coli is an FAD-containing oxidoreductase that transports electrons between flavodoxin or ferredoxin and NADPH. Together with flavodoxin, the enzyme is involved in the reductive activation of three E. coli enzymes: cobalamin-dependent methionine synthase, pyruvate formate lyase and anaerobic ribonucleotide reductase. An additional function for the oxidoreductase appears to be to protect the bacteria against oxygen radicals. The three-dimensional structure of flavodoxin reductase has been solved by multiple isomorphous replacement, and has been refined at 1.7 Å to an R-value of 18.4% and R free 24.8%. The monomeric molecule contains one β-sandwich FAD domain and an α/β NADP domain. The overall structure is similar to other reductases of the NADP-ferredoxin reductase family in spite of the low sequence similarities within the family. Flavodoxin reductase lacks the loop which is involved in the binding of the adenosine moiety of FAD in other FAD binding enzymes of the superfamily but is missing in the FMN binding phthalate dioxygenase reductase. Instead of this loop, the adenine interacts with an extra tryptophan at the C terminus. The FAD in flavodoxin reductase has an unusual bent conformation with a hydrogen bond between the adenine and the isoalloxazine. This is probably the cause of the unusual spectrum of the enzyme. There is a pronounced cleft close to the isoalloxazine that appears to be well suited for binding of flavodoxin/ferredoxin. Two extra short strands of the NADP-binding domain probably act as an anchor point for the binding of flavodoxin.

Electrostatic properties deduced from refined structures of NADH-cytochrome b5 reductase and the other flavin-dependent reductases: pyridine nucleotide-binding and interaction with an electron-transfer partner.

Electrostatic properties on the protein surface were examined on the basis of the crystal structure of NADH-cytochrome b5 reductase refined to a crystallographic R factor of 0.223 at 2.1 A resolution and of the other three flavin-dependent reductases. A structural comparison of NADH-cytochrome b5 reductase with the other flavin-dependent reductases, ferredoxin-NADP+ reductase, phthalate dioxygenase reductase, and nitrate reductase, showed that the alpha/beta structure is the common motif for binding pyridine nucleotide. Although the amino acid residues associated with pyridine nucleotide-binding are not conserved, the electrostatic properties and the location of the pyridine nucleotide-binding pockets of NADH-requiring reductases were similar to each other. The electrostatic potential of the surface near the flavin-protruding side (dimethylbenzene end of the flavin ring) of NADH-cytochrome b5 reductase was positive over a wide area while that of the surface near the heme-binding site of cytochrome b5 was negative. This implied that the flavin-protruding side of NADH-cytochrome b5 reductase is suitable for interacting with its electron-transfer partner, cytochrome b5. This positive potential area is conserved among four flavin-dependent reductases. A comparison of the electron-transfer partners of four flavin-dependent reductases showed that there are significant differences in the distribution of electrostatic potential between inter-molecular and inter-domain electron-transfer reactions.

A chimeric iron-sulfur flavoprotein endowed with NADPH-cytochrome c reductase activity.

Ferredoxin-NADP' reductase (FNR) catalyses the last step in the photosynthetic electron transport chain: it pairs single electrons carried by ferredoxin (Fd) and transfers them as a hydride to NADP'. FNR is the prototype of a large family of flavin-dependent enzymes that function in various electron transport chains as transducers between oneand two-electron carriers [ 1, 21. Besides the flavin prosthetic group, these enzymes have additional reducible cofactors, which can reside in linked domains or in dissociable protein units, as is the case for the FNRiFd couple. FNR and Fd contain FAD and a [2Fe-2S] cluster, respectively. Both proteins from spinach leaves have been cloned and expressed in E. coli [3, 41. The three-dimensional structures of spinach FNR [5] and of cyanobacterial Fd are known at high resolution. To provide a new tool for studying the interaction between Fd and FNR, a chimeric protein, comprising the two functional units on the same polypeptide chain, was designed. The chimeric protein should acquire a domain organisation resembling that of known members of the family, such as phthalate dioxygenase reductase and oxygenase reductase, where a Fd-like domain is linked to the basic FNR twodomain motif [4]. A gene construct encoding the chimeric three-domain protein has been obtained by fusion of the 5' end of the cDNA part coding for the mature spinach FNR to the 3' end of the cDNA fragment encoding the mature isoform I of spinach leaf Fd (Fd I). This arrangement was chosen to take advantage of the fact that the first 18 residues of FNR do not have an ordered structure, thus providing a natural spacer arm between the folding units. This construct has been inserted in the 'vector PET-lld and overexpressed in E. coli. The recombinant protein was synthesised in the bacterial host at a level of about 1% of the soluble proteins. The chimera has been purified with an overall yield of about 45% by a combination of the procedures used to isolate the individual proteins. In non-denaturing PAGE, the fusion protein showed an electrophoretic mobility intermediate between those of FNR and Fd I. The apparent M, of the chimeric protein, as measured by 0.4 L I i i 1 i I I I I i I I I I I I i i I 1 1

Structure and mechanism of the iron-sulfur flavoprotein phthalate dioxygenase reductase.

Transfer of electrons between pyridine nucleotides (obligatory two-electron carriers) and hemes or [2Fe-2S] centers (obligatory one-electron carriers) is an essential step mediated by flavins in respiration, photosynthesis, and many oxygenase systems. Phthalate dioxygenase reductase (PDR), a soluble iron-sulfur flavoprotein from Pseudomonas cepacia, is a convenient model for the study of this type of electron transfer. PDR is folded into three domains; the NH2-terminal FMN binding and central NAD(H) binding domains are closely related to ferredoxin-NADP+ reductase (FNR). The COOH-terminal [2Fe-2S] domain is similar to plant ferredoxins, and can be removed by proteolysis without significantly altering the reactivity of the FNR-like domains. Kinetic studies have identified sequential steps in the reaction of PDR with NADH that involve pyridine nucleotide binding, hydride transfer to FMN, and intramolecular electron transfer from the reduced flavin to the [2Fe-2S] cluster. Crystal structures of reduced and liganded PDR correspond to some of the intermediates formed during reduction by NADH. Small structural changes that are observed in the vicinity of the cofactors upon reduction or NAD(H) binding may provide part of the reorganization energy or contribute to the gating mechanism that controls intramolecular electron transfer.

Specific arrangement of three amino acid residues for flavin-binding barrel structures in NADH-cytochrome b5 reductase and the other flavin-dependent reductases

The structure of NADH-cytochrome b 5 reductase from pig liver microsomes has been refined to a crystallographic R factor of 0.223 at 2.4 Å resolution. A structural comparison between the flavin-binding β barrel domain of NADH-cytochrome b 5 reductase and those of the other flavin-dependent reductases, ferredoxin-NADP + reductase, phthalate dioxygenase reductase and nitrate reductase, indicated that the overall barrel foldings are similar to each other and that the specific arrangement of three amino acid residues (Arg, Tyr and Ser/Thr) is usually necessary for flavin-binding. These conserved residues overlap each other in their three-dimensional structures and stabilize the flavin-binding site in the four flavin-dependent reductases.

Evidence for a Magic Magnetic Configuration between FMN and the [2Fe-2S]+ Center of Phthalate Dioxygenase Reductase of Pseudomonas cepacia

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTEvidence for a Magic Magnetic Configuration between FMN and the [2Fe-2S]+ Center of Phthalate Dioxygenase Reductase of Pseudomonas cepaciaPatrick Bertrand, Claude More, and Philippe CamensuliCite this: J. Am. Chem. Soc. 1995, 117, 6, 1807–1809Publication Date (Print):February 1, 1995Publication History Published online1 May 2002Published inissue 1 February 1995https://doi.org/10.1021/ja00111a020RIGHTS & PERMISSIONSArticle Views77Altmetric-Citations16LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (352 KB) Get e-Alerts Get e-Alerts

Reaction of Phthalate Dioxygenase Reductase with NADH and NAD: Kinetic and Spectral Characterization of Intermediates

Phthalate dioxygenase reductase (PDR) is an electron transferase that contains FMN, which accepts a hydride from NADH, and a [2Fe-2S] center, which transfers electrons to phthalate dioxygenase. The reduction of PDR by NADH has been studied by stopped-flow spectroscopy. Data from studies using both portio- and deuterio-NADH were analyzed by nonlinear curve fitting and numerical simulation techniques. The results of these analyses indicate that the reductive half-reaction of PDR consists of five distinct kinetic phases: (a) NADH binds to form a primary Michaelis complex (MC-1) (Kd = 50 microM). (b) The enzyme undergoes a structural change (116 +/- 5 s-1) resulting in a charge-transfer complex (CT-1). (c) The next phase in the reaction shows a deuterium isotope effect of 7.0 when (4R)-[2H]NADH (NADD) is substituted for NADH, identifying this step as the one involving hydride transfer. The rate of hydride transfer from NADH to FMN is 70 s-1, and this process results in a charge-transfer intermediate between the flavin hydroquinone anion and NAD (CT). (d) Internal electron transfer from the flavin to the iron-sulfur center, which is only 35 +/- 4 s-1, then results in an intermediate consisting of a reduced [2Fe-2S] center and a neutral flavin semiquinone (SQ). It is surprising that this rate is so slow, since the shortest interatomic distance between these centers is only 4.7 A [Correll, C. C., et al. (1992) Science 258, 1604-1610]. The 2-electron-reduced form of PDR (SQ in Figure 1) binds weakly to the reaction product, NAD (Kd = 3.7 mM), but forms a tight complex with NADH (Kd = 10 microM). (e) Two molecules of the reduced iron-sulfur flavin semiquinone (SQ) form of PDR then undergo a relatively slow second-order disproportionation reaction, resulting in one molecule of 3-electron-reduced PDR and one molecule of 1-electron-reduced PDR. The latter reacts rapidly with excess NADH to form a 3-electron-reduced PDR.

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 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 beta-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.