Spectroscopic studies and characterization of a novel electron-transfer chain from Escherichia coli involving a flavorubredoxin and its flavoprotein reductase partner

A novel two-component enzyme system from Escherichia coli involving a flavorubredoxin (FlRd) and its reductase was studied in terms of spectroscopic, redox, and biochemical properties of its constituents. FlRd contains one FMN and one rubredoxin (Rd) center per monomer. To assess the role of the Rd domain, FlRd and a truncated form lacking the Rd domain (FlRdDeltaRd), were characterized. FlRd contains 2.9+/-0.5 iron atoms/subunit, whereas FlRdDeltaRd contains 2.1+/-0.6 iron atoms/subunit. While for FlRd one iron atom corresponds to the Rd center, the other two irons, also present in FlRdDeltaRd, are most probably due to a di-iron site. Redox titrations of FlRd using EPR and visible spectroscopies allowed us to determine that the Rd site has a reduction potential of -140+/-15 mV, whereas the FMN undergoes reduction via a red-semiquinone, at -140+/-15 mV (Fl(ox)/Fl(sq)) and -180+/-15 mV (Fl(sq)/Fl(red)), at pH 7.6. The Rd site has the lowest potential ever reported for a Rd center, which may be correlated with specific amino acid substitutions close to both cysteine clusters. The gene adjacent to that encoding FlRd was found to code for an FAD-containing protein, (flavo)rubredoxin reductase (FlRd-reductase), which is capable of mediating electron transfer from NADH to Desulfovibrio gigas Rd as well as to E. coli FlRd. Furthermore, electron donation was found to proceed through the Rd domain of FlRd as the Rd-truncated protein does not react with FlRd-reductase. In vitro, this pathway links NADH oxidation with dioxygen reduction. The possible function of this chain is discussed considering the presence of FlRd homologues in all known genomes of anaerobes and facultative aerobes.     

Kinetics of electron transfer from NADH to the Escherichia coli nitric oxide reductase flavorubredoxin

Escherichia coli flavorubredoxin (FlRd) belongs to the family of flavodiiron proteins (FDPs), microbial enzymes that are expressed to scavenge nitric oxide (NO) under anaerobic conditions. To degrade NO, FlRd has to be reduced by NADH via the FAD-binding protein flavorubredoxin reductase, thus the kinetics of electron transfer along this pathway was investigated by stopped-flow absorption spectroscopy. We found that NADH, but not NADPH, quickly reduces the FlRd-reductase (k = 5.5 +/- 2.2 x 10(6) M(-1).s(-1) at 5 degrees C), with a limiting rate of 255 +/- 17 s(-1). The reductase in turn quickly reduces the rubredoxin (Rd) center of FlRd, as assessed at 5 degrees C working with the native FlRd enzyme (k = 2.4 +/- 0.1 x 10(6) m(-1).s(-1)) and with its isolated Rd-domain (k approximately 1 x 10(7) M(-1).s(-1)); in both cases the reaction was found to be dependent on pH and ionic strength. In FlRd the fast reduction of the Rd center occurs synchronously with the formation of flavin mononucleotide semiquinone. Our data provide evidence that (a) FlRd-reductase rapidly shuttles electrons between NADH and FlRd, a prerequisite for NO reduction in this detoxification pathway, and (b) the electron accepting site in FlRd, the Rd center, is in very fast redox equilibrium with the flavin mononucleotide.     

Quaternary structure of flavorubredoxin as revealed by synchrotron radiation small-angle X-ray scattering

Flavodiiron proteins (FDP) are modular enzymes which function as NO and/or O(2) reductases. Although the majority is composed of two structural domains, the homolog found in Escherichia coli, flavorubredoxin, possesses an extra C-terminal module consisting of a linker and a rubredoxin (Rd) domain necessary for interprotein redox processes. In order to investigate the location of the Rd domain with respect to the flavodiiron structural core, small-angle X-ray scattering was used to construct low-resolution structural models of flavorubredoxin. Scattering patterns from the Rd domain, the FDP core, and full-length flavorubredoxin were collected. The latter two species were found to be tetrameric in solution. Ab initio shape reconstruction and rigid-body modeling indicate a peripheral location for the Rd domains, which appear to have weak contacts with the FDP core. This finding suggests that Rd behaves as an independent domain and is freely available to participate in redox reactions with protein partners.     

Crystal structure of rubredoxin from Desulfovibrio gigas to ultra-high 0.68 A resolution

Rubredoxin (D.g. Rd) is a small non-heme iron-sulfur protein shown to function as a redox coupling protein from the sulfate reducing bacteria Desulfovibrio gigas. The protein is generally purified from anaerobic bacteria in which it is thought to be involved in electron transfer or exchange processes. Rd transfers an electron to oxygen to form water as part of a unique electron transfer chain, composed by NADH:rubredoxin oxidoreductase (NRO), rubredoxin and rubredoxin:oxygen oxidoreductase (ROO) in D.g. The crystal structure of D.g. Rd has been determined by means of both a Fe single-wavelength anomalous dispersion (SAD) signal and the direct method, and refined to an ultra-high 0.68 A resolution, using X-ray from a synchrotron. Rd contains one iron atom bound in a tetrahedral coordination by the sulfur atoms of four cysteinyl residues. Hydrophobic and pi-pi interactions maintain the internal Rd folding. Multiple conformations of the iron-sulfur cluster and amino acid residues are observed and indicate its unique mechanism of electron transfer. Several hydrogen bonds, including N-H...SG of the iron-sulfur, are revealed clearly in maps of electron density. Abundant waters bound to C-O peptides of residues Val8, Cys9, Gly10, Ala38, and Gly43, which may be involved in electron transfer. This ultrahigh-resolution structure allows us to study in great detail the relationship between structure and function of rubredoxin, such as salt bridges, hydrogen bonds, water structures, cysteine ligands, iron-sulfur cluster, and distributions of electron density among activity sites. For the first time, this information will provide a clear role for this protein in a strict anaerobic bacterium.     

Isolation and characterization of rubrerythrin, a non-heme iron protein from Desulfovibrio vulgaris that contains rubredoxin centers and a hemerythrin-like binuclear iron cluster

A new non-heme iron protein from the periplasmic fraction of Desulfovibrio vulgaris (Hildenbourough NCIB 8303) has been purified to homogeneity, and its amino acid composition, molecular weight, redox potential, iron content, and optical, EPR, and M?ssbauer spectroscopic properties have been determined. This new protein is composed of two identical subunits with subunit molecular weight of 21,900 and contains four iron atoms per molecule. The as-purified oxidized protein exhibits an optical spectrum with absorption maxima at 492, 365, and 280 nm, and its EPR spectrum shows resonances at g = 4.3 and 9.4, characteristic of oxidized rubredoxin. The M?ssbauer data indicate the presence of approximately equal amounts of two types of iron; we named them the Rd-like and the Hr-like iron due to their similarity to the iron centers of rubredoxins (Rds) and hemerythrins (Hrs), respectively. For the Rd-like iron, the measured fine and hyperfine parameters (D = 1.5 cm-1, E/D = 0.26, delta EQ = -0.55 mm/s, delta = 0.27 mm/s, Axx/gn beta n = -16.5 T, Ayy/gn beta n = -15.6 T, and Azz/gn beta n = -17.0 T) are almost identical with those obtained for the rubredoxin from Clostridium pasteurianum. Redox-titration studies monitored by EPR, however, showed that these Rd-like centers have a midpoint redox potential of +230 +/- 10 mV, approximately 250 mV more positive than those reported for rubredoxins. Another unusual feature of this protein is the presence of the Hr-like iron atoms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Iron-based redox centres of reductase and oxygenase components of phenol hydroxylase from A. radioresistens: a redox chain working at highly positive redox potentials

This is the first report of the direct electrochemistry of the reductase (PHR) and oxygenase (PHO) components of phenol hydroxylase from Acinetobacter radioresistens S13 studied by cyclic and differential pulse voltammetry. The PHR contains one 2Fe2S cluster and one FAD that mediate the transfer of electrons from NAD(P)H to the non-heme diiron cluster of PHO. Cyclic and differential pulse voltammetry (CV and DPV) on glassy carbon showed two redox pairs with midpoint potentials at +131.5 ± 13 mV and -234 ± 3 mV versus normal hydrogen electrode (NHE). The first redox couple is attributed to the FeS centre, while the second one corresponds to free FAD released by the protein. DPV scans on native and guanidinium chloride treated PHR highlighted the presence of a split signal (ΔE ≈ 100 mV) attributed to heterogeneous properties of the 2Fe2S cluster interacting with the electrode, possibly due to the presence of two protein conformers and consistently with the large peak-to-peak separation and the peak broadening observed in CV. DPV experiments on gold electrodes performed on PHO confirm a consistently higher reduction potential at +396 mV vs. NHE. The positive redox potentials measured by direct electrochemistry for the FeS cluster in PHR and for the non-heme diiron cluster of PHO show that the entire phenol hydroxylase system works at higher potentials than those reported for structurally similar enzymes, for example methane monooxygenases.     

A coupled dinuclear iron cluster that is perturbed by substrate binding in myo-inositol oxygenase

myo-Inositol oxygenase (MIOX) uses iron as its cofactor and dioxygen as its cosubstrate to effect the unique, ring-cleaving, four-electron oxidation of its cyclohexan-(1,2,3,4,5,6-hexa)-ol substrate to d-glucuronate. The nature of the iron cofactor and its interaction with the substrate, myo-inositol (MI), have been probed by electron paramagnetic resonance (EPR) and M?ssbauer spectroscopies. The data demonstrate the formation of an antiferromagnetically coupled, high-spin diiron(III/III) cluster upon treatment of solutions of Fe(II) and MIOX with excess O(2) or H(2)O(2) and the formation of an antiferromagnetically coupled, valence-localized, high-spin diiron(II/III) cluster upon treatment with either limiting O(2) or excess O(2) in the presence of a mild reductant (e.g., ascorbate). Marked changes to the spectra of both redox forms upon addition of MI and analogy to changes induced by binding of phosphate to the diiron(II/III) cluster of the protein phosphatase, uteroferrin, suggest that MI coordinates directly to the diiron cluster, most likely in a bridging mode. The addition of MIOX to the growing family of non-heme diiron oxygenases expands the catalytic range of the family beyond the two-electron oxidation (hydroxylation and dehydrogenation) reactions catalyzed by its more extensively studied members such as methane monooxygenase and stearoyl acyl carrier protein Delta(9)-desaturase.     

X-ray crystal structures of Moorella thermoacetica FprA. Novel diiron site structure and mechanistic insights into a scavenging nitric oxide reductase

Several members of a widespread class of bacterial and archaeal metalloflavoproteins, called FprA, likely function as scavenging nitric oxide reductases (S-NORs). However, the only published X-ray crystal structure of an FprA is for a protein characterized as a rubredoxin:dioxygen oxidoreductase (ROO) from Desulfovibrio gigas. Therefore, the crystal structure of Moorella thermoacetica FprA, which has been established to function as an S-NOR, was solved in three different states: as isolated, reduced, and reduced, NO-reacted. As is the case for D. gigas ROO, the M. thermoacetica FprA contains a solvent-bridged non-heme, non-sulfur diiron site with five-coordinate iron centers bridged by an aspartate, and terminal glutamate, aspartate, and histidine ligands. However, the M. thermoacetica FprA diiron site showed four His ligands, two to each iron, in all three states, whereas the D. gigas ROO diiron site was reported to contain only three His ligands, even though the fourth His residue is conserved. The Fe1-Fe2 distance within the diiron site of M. thermoacetica FprA remained at 3.2-3.4 A with little or no movement of the protein ligands in the three different states and with conservation of the two proximal open coordination sites. Molecular modeling indicated that each open coordination site can accommodate an end-on NO. This relatively rigid and symmetrical diiron site structure is consistent with formation of a diferrous dinitrosyl as the committed catalytic intermediate leading to formation of N(2)O. These results provide new insight into the structural features that fine-tune biological non-heme diiron sites for dioxygen activation vs nitric oxide reduction.     

Acceptor side effects on the electron transfer at cryogenic temperatures in intact photosystem II

In intact PSII, both the secondary electron donor (Tyr(Z)) and side-path electron donors (Car/Chl(Z)/Cyt(b)(559)) can be oxidized by P(680)(+) at cryogenic temperatures. In this paper, the effects of acceptor side, especially the redox state of the non-heme iron, on the donor side electron transfer induced by visible light at cryogenic temperatures were studied by EPR spectroscopy. We found that the formation and decay of the S(1)Tyr(Z) EPR signal were independent of the treatment of K(3)Fe(CN)(6), whereas formation and decay of the Car(+)/Chl(Z)(+) EPR signal correlated with the reduction and recovery of the Fe(3+) EPR signal of the non-heme iron in K(3)Fe(CN)(6) pre-treated PSII, respectively. Based on the observed correlation between Car/Chl(Z) oxidation and Fe(3+) reduction, the oxidation of non-heme iron by K(3)Fe(CN)(6) at 0 degrees C was quantified, which showed that around 50-60% fractions of the reaction centers gave rise to the Fe(3+) EPR signal. In addition, we found that the presence of phenyl-p-benzoquinone significantly enhanced the yield of Tyr(Z) oxidation. These results indicate that the electron transfer at the donor side can be significantly modified by changes at the acceptor side, and indicate that two types of reaction centers are present in intact PSII, namely, one contains unoxidizable non-heme iron and another one contains oxidizable non-heme iron. Tyr(Z) oxidation and side-path reaction occur separately in these two types of reaction centers, instead of competition with each other in the same reaction centers. In addition, our results show that the non-heme iron has different properties in active and inactive PSII. The oxidation of non-heme iron by K(3)Fe(CN)(6) takes place only in inactive PSII, which implies that the Fe(3+) state is probably not the intermediate species for the turnover of quinone reduction.     

The O2-scavenging flavodiiron protein in the human parasite Giardia intestinalis

The flavodiiron proteins (FDP) are widespread among strict or facultative anaerobic prokaryotes, where they are involved in the response to nitrosative and/or oxidative stress. Unexpectedly, FDPs were fairly recently identified in a restricted group of microaerobic protozoa, including Giardia intestinalis, the causative agent of the human infectious disease giardiasis. The FDP from Giardia was expressed, purified, and extensively characterized by x-ray crystallography, stopped-flow spectroscopy, respirometry, and NO amperometry. Contrary to flavorubredoxin, the FDP from Escherichia coli, the enzyme from Giardia has high O(2)-reductase activity (>40 s(-1)), but very low NO-reductase activity (approximately 0.2 s(-1)); O(2) reacts with the reduced protein quite rapidly (milliseconds) and with high affinity (K(m) < or = 2 microM), producing H(2)O. The three-dimensional structure of the oxidized protein determined at 1.9A resolution shows remarkable similarities with prokaryotic FDPs. Consistent with HPLC analysis, the enzyme is a dimer of dimers with FMN and the non-heme di-iron site topologically close at the monomer-monomer interface. Unlike the FDP from Desulfovibrio gigas, the residue His-90 is a ligand of the di-iron site, in contrast with the proposal that ligation of this histidine is crucial for a preferential specificity for NO. We propose that in G. intestinalis the primary function of FDP is to efficiently scavenge O(2), allowing this microaerobic parasite to survive in the human small intestine, thus promoting its pathogenicity.     

Determinants of protein hyperthermostability: purification and amino acid sequence of rubredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus and secondary structure of the zinc adduct by NMR

The purification, amino acid sequence, and two-dimensional 1H NMR results are reported for the rubredoxin (Rd) from the hyperthermophilic archaebacterium Pyrococcus furiosus, an organism that grows optimally at 100 degrees C. The molecular mass (5397 Da), iron content (1.2 +/- 0.2 g-atom of Fe/mol), UV-vis spectrophotometric properties, and amino acid sequence (60% sequence identity with Clostridium pasteurianum Rd) are found to be typical of this class of redox protein. However, P. furiosus Rd is remarkably thermostable, being unaffected after incubation for 24 h at 95 degrees C. One- and two-dimensional 1H nuclear magnetic resonance spectra of the oxidized [Fe(III)Rd] and reduced [Fe(II)Rd] forms of P. furiosus Rd exhibited substantial paramagnetic line broadening, and this precluded detailed 3D structural studies. The apoprotein was not readily amenable to NMR studies due to apparent protein oxidation involving the free cysteine sulfhydryls. However, high-quality NMR spectra were obtained for the Zn-substituted protein, Zn(Rd), enabling detailed NMR signal assignment for all backbone amide and alpha and most side-chain protons. Secondary structural elements were determined from qualitative analysis of 2D Overhauser effect spectra. Residues A1-K6, Y10-E14, and F48-E51 form a three-strand antiparallel beta-sheet, which comprises ca. 30% of the primary sequence. Residues C5-Y10 and C38-A43 form types I and II amide-sulfur tight turns common to iron-sulfur proteins. These structural elements are similar to those observed by X-ray crystallography for native Rd from the mesophile C. pasteurianum. However, the beta-sheet domain in P. furiosus Rd is larger than that in C. pasteurianum Rd and appears to begin at the N-terminal residue. From analysis of the secondary structure, potentially stabilizing electrostatic interactions involving the charged groups of residues Ala(1), Glu(14), and Glu(52) are proposed. These interactions, which are not present in rubredoxins from mesophilic organisms, may prevent the beta-sheet from "unzipping" at elevated temperatures.     

Direct electrochemistry of porcine purple acid phosphatase (uteroferrin)

Cyclic voltammetry of the non-heme diiron enzyme porcine purple acid phosphatase (uteroferrin, Uf) has been reported for the first time. Totally reversible one-electron oxidation responses (FeIII-FeII --> FeII-FeIII) are seen both in the absence and in the presence of weak competitive inhibitors phosphate and arsenate, and dissociation constants of these oxoanion complexes formed with uteroferrin in its oxidized state (Uf(o)) have been determined. The effect of pH on the redox potentials has been investigated in the range 3 < pH < 6.5, enabling acid dissociation constants for Uf(o) and its phosphate and arsenate complexes to be calculated.     

An EPR study of the dinuclear iron site in the soluble methane monooxygenase from Methylococcus capsulatus (Bath) reduced by one electron at 77 K: the effects of component interactions and the binding of small molecules to the diiron(III) center

Reduction of the soluble methane monooxygenase hydroxylase (MMOH) from Methylococcus capsulatus (Bath) in frozen 4:1 buffer/glycerol solutions at 77 K by mobile electrons generated by gamma-irradiation produces an EPR-detectable, mixed-valent Fe(II)Fe(III) center. At this temperature the conformation of the enzyme remains essentially unaltered during reduction, so the mixed-valent EPR spectra serve to probe the active site structure of the EPR-silent, diiron(III) state. The EPR spectra of the cryoreduced samples reveal that the diiron(III) cluster of the resting hydroxylase has at least two chemically distinct forms, the structures of which differ from that of the equilibrium Fe(II)Fe(III) site. Their relative populations depend on pH, the presence of component B, and formation of the MMOH/MMOB complex by reoxidation of the reduced, diiron(II) hydroxylase. The formation of complexes between MMOB, MMOR, and the oxidized hydroxylase does not measurably affect the structure of the diiron(III) site. Cryogenic reduction in combination with EPR spectroscopy has also provided information about interaction of MMOH in the diiron(III) state with small molecules. The diiron(III) center binds methanol and phenols, whereas DMSO and methane have no measurable effect on the EPR properties of cryoreduced hydroxylase. Addition of component B favors the binding of some exogenous ligands, such as DMSO and glycerol, to the active site diiron(III) state and markedly perturbs the structure of the diiron(III) cluster complexed with methanol or phenol. The results reveal different reactivity of the Fe(III)Fe(III) and Fe(II)Fe(III) redox states of MMOH toward exogenous ligands. Moreover, unlike oxidized hydroxylase, the binding of exogenous ligands to the protein in the mixed-valent state is allosterically inhibited by MMOB. The differential reactivity of the hydroxylase in its diiron(III) and mixed-valent states toward small molecules, as well as the structural basis for the regulatory effects of component B, is interpreted in terms of a model involving carboxylate shifts of a flexible glutamate ligand at the Fe(II)Fe(III) center.     

Leucine 41 is a gate for water entry in the reduction of Clostridium pasteurianum rubredoxin

Biological electron transfer is an efficient process even though the distances between the redox moieties are often quite large. It is therefore of great interest to gain an understanding of the physical basis of the rates and driving forces of these reactions. The structural relaxation of the protein that occurs upon change in redox state gives rise to the reorganizational energy, which is important in the rates and the driving forces of the proteins involved. To determine the structural relaxation in a redox protein, we have developed methods to hold a redox protein in its final oxidation state during crystallization while maintaining the same pH and salt conditions of the crystallization of the protein in its initial oxidation state. Based on 1.5 A resolution crystal structures and molecular dynamics simulations of oxidized and reduced rubredoxins (Rd) from Clostridium pasteurianum (Cp), the structural rearrangements upon reduction suggest specific mechanisms by which electron transfer reactions of rubredoxin should be facilitated. First, expansion of the [Fe-S] cluster and concomitant contraction of the NH...S hydrogen bonds lead to greater electrostatic stabilization of the extra negative charge. Second, a gating mechanism caused by the conformational change of Leucine 41, a nonpolar side chain, allows transient penetration of water molecules, which greatly increases the polarity of the redox site environment and also provides a source of protons. Our method of producing crystals of Cp Rd from a reducing solution leads to a distribution of water molecules not observed in the crystal structure of the reduced Rd from Pyrococcus furiosus. How general this correlation is among redox proteins must be determined in future work. The combination of our high-resolution crystal structures and molecular dynamics simulations provides a molecular picture of the structural rearrangement that occurs upon reduction in Cp rubredoxin.     

Protein control of electron transfer rates via polarization: molecular dynamics studies of rubredoxin

The protein matrix of an electron transfer protein creates an electrostatic environment for its redox site, which influences its electron transfer properties. Our studies of Fe-S proteins indicate that the protein is highly polarized around the redox site. Here, measures of deviations of the environmental electrostatic potential from a simple linear dielectric polarization response to the magnitude of the charge are proposed. In addition, a decomposition of the potential is proposed here to describe the apparent deviations from linearity, in which it is divided into a "permanent" component that is independent of the redox site charge and a dielectric component that linearly responds or polarizes to the charge. The nonlinearity measures and the decomposition were calculated for Clostridium pasteurianum rubredoxin from molecular dynamics simulations. The potential in rubredoxin is greater than expected from linear response theory, which implies it is a better electron acceptor than a redox site analog in a solvent with a dielectric constant equivalent to that of the protein. In addition, the potential in rubredoxin is described well by a permanent potential plus a linear response component. This permanent potential allows the protein matrix to create a favorable driving force with a low activation barrier for accepting electrons. The results here also suggest that the reduction potential of rubredoxin is determined mainly by the backbone and not the side chains, and that the redox site charge of rubredoxin may help to direct its folding.     

A role for rubredoxin in oxidative stress protection in Desulfovibrio vulgaris: catalytic electron transfer to rubrerythrin and two-iron superoxide reductase

Desulfovibrio vulgaris rubredoxin, which contains a single [Fe(SCys)4] site, is shown to be a catalytically competent electron donor to two enzymes from the same organism, namely, rubrerythrin and two-iron superoxide reductase (a.k.a. rubredoxin oxidoreductase or desulfoferrodoxin). These two enzymes have been implicated in catalytic reduction of hydrogen peroxide and superoxide, respectively, during periods of oxidative stress in D. vulgaris, but their proximal electron donors had not been characterized. We further demonstrate the incorrectness of a previous report that rubredoxin is not an electron donor to the superoxide reductase and describe convenient assays for demonstrating the catalytic competence of all three proteins in their respective functions. Rubrerythrin is shown to be an efficient rubredoxin peroxidase in which the rubedoxin:hydrogen peroxide redox stoichiometry is 2:1 mol:mol. Using spinach ferredoxin-NADP+ oxidoreductase (FNR) as an artificial, but proficient, NADPH:rubredoxin reductase, rubredoxin was further found to catalyze rapid and complete reduction of all Fe3+ to Fe2+ in rubrerythrin by NADPH under anaerobic conditions. The combined system, FNR/rubredoxin/rubrerythrin, was shown to function as a catalytically competent NADPH peroxidase. Another small rubredoxin-like D. vulgaris protein, Rdl, could not substitute for rubredoxin as a peroxidase substrate of rubrerythrin. Similarly, D. vulgaris rubredoxin was demonstrated to efficiently catalyze reduction of D. vulgaris two-iron superoxide reductase and, when combined with FNR, to function as an NADPH:superoxide oxidoreductase. We suggest that, during periods of oxidative stress, rubredoxin could divert electron flow from the electron transport chain of D. vulgaris to rubrerythrin and superoxide reductase, thereby simultaneously protecting autoxidizable redox enzymes and lowering intracellular hydrogen peroxide and superoxide levels.     

In vitro reconstitution of an NADPH-dependent superoxide reduction pathway from Pyrococcus furiosus

A scheme for the detoxification of superoxide in Pyrococcus furiosus has been previously proposed in which superoxide reductase (SOR) reduces (rather than dismutates) superoxide to hydrogen peroxide by using electrons from reduced rubredoxin (Rd). Rd is reduced with electrons from NAD(P)H by the enzyme NAD(P)H:rubredoxin oxidoreductase (NROR). The goal of the present work was to reconstitute this pathway in vitro using recombinant enzymes. While recombinant forms of SOR and Rd are available, the gene encoding P. furiosus NROR (PF1197) was found to be exceedingly toxic to Escherichia coli, and an active recombinant form (rNROR) was obtained via a fusion protein expression system, which produced an inactive form of NROR until cleavage. This allowed the complete pathway from NAD(P)H to the reduction of SOR via NROR and Rd to be reconstituted in vitro using recombinant proteins. rNROR is a 39.9-kDa protein whose sequence contains both flavin adenine dinucleotide (FAD)- and NAD(P)H-binding motifs, and it shares significant similarity with known and putative Rd-dependent oxidoreductases from several anaerobic bacteria, both mesophilic and hyperthermophilic. FAD was shown to be essential for activity in reconstitution assays and could not be replaced by flavin mononucleotide (FMN). The bound FAD has a midpoint potential of -173 mV at 23 degrees C (-193 mV at 80 degrees C). Like native NROR, the recombinant enzyme catalyzed the NADPH-dependent reduction of rubredoxin both at high (80 degrees C) and low (23 degrees C) temperatures, consistent with its proposed role in the superoxide reduction pathway. This is the first demonstration of in vitro superoxide reduction to hydrogen peroxide using NAD(P)H as the electron donor in an SOR-mediated pathway.