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Appl Environ Microbiol. Mar 2005; 71(3): 1522–1530.
PMCID: PMC1065123

In Vitro Reconstitution of an NADPH-Dependent Superoxide Reduction Pathway from Pyrococcus furiosus

Abstract

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°C (−193 mV at 80°C). Like native NROR, the recombinant enzyme catalyzed the NADPH-dependent reduction of rubredoxin both at high (80°C) and low (23°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.

Studies of hydrothermal vent systems have shown that anaerobic hyperthermophilic archaea such as Pyrococcus furiosus can be exposed to significant levels of oxygen at low temperatures when hot, anaerobic vent fluids mix with cold, oxygen-saturated seawater (15, 20, 40). Such oxygen exposure might be expected to incur serious cellular damage, if not cell death, as anaerobes do not typically contain the classical superoxide dismutase and catalase enzymes with which to detoxify oxygen and reactive oxygen species (ROS) (10, 24, 25, 37). However, it has recently been proposed that anaerobes such as P. furiosus contain an oxygen detoxification system that does not use superoxide dismutase and catalase (22). Instead, they employ a novel enzyme system centered around superoxide reductase (SOR), which functions to reduce superoxide to hydrogen peroxide (4, 22). Unlike the conversion of superoxide to hydrogen peroxide by superoxide dismutase, the reduction of superoxide by SOR produces hydrogen peroxide but no oxygen, which is obviously advantageous to anaerobic organisms. It is thought that the hydrogen peroxide produced by the reduction of superoxide can be removed through reduction by enzymes such as peroxiredoxin and NADH peroxidase (22, 36, 41). More recently, rubrerythrin (30), another nonheme iron-containing protein unique to anaerobes, has been shown to function as an NADH-dependent peroxidase in P. furiosus (42). Subsequent studies have shown that the SOR-related detoxification system is also present in Archaeoglobus fulgidus, Desulfovibrio gigas, Desulfoarculus baarsii, and Treponema pallidum (1, 23, 28, 29, 33, 39). In fact, analysis of anaerobes and microaerophiles with sequenced genomes shows that homologs of SOR are present in all cases (3, 4).

For the superoxide reduction process, it was proposed (22) that rubredoxin, a small (6-kDa) iron-containing protein (8), serves as the electron donor in P. furiosus. Rubredoxin was proposed to be reduced in vivo by NAD(P)H:rubredoxin oxidoreductase (NROR) due to the high specificity of this reaction in vitro (31). NROR has been purified from P. furiosus as a monomeric, flavin adenine dinucleotide (FAD)-containing protein of 45 kDa (31). Intriguingly, like SOR, NROR is enzymatically active in vitro at temperatures (23°C) that are far below the optimal growth temperature of P. furiosus (100°C) (14). This property is highly unusual for enzymes that have been characterized from hyperthermophilic sources (6, 22, 31). In fact, the ability of these two enzymes to function at low temperature supports the notion of their roles in oxygen protection, as P. furiosus can be exposed to significant levels of oxygen at low temperature (20, 40).

It has yet to be demonstrated, however, that NAD(P)H can serve as an electron donor ultimately for superoxide reduction by P. furiosus SOR. Although recombinant forms of SOR (11) and rubredoxin (21) are available for in vitro assays, only limited amounts of NROR are obtained from P. furiosus biomass (31). The objectives of the present study were, therefore, to obtain the recombinant form of P. furiosus NROR, to reconstitute an in vitro recombinant superoxide reduction pathway, and to demonstrate that NADPH can serve as an electron source for superoxide reduction to peroxide. However, our preliminary recombinant expression attempts showed that the gene encoding P. furiosus NROR (PF1197) was exceedingly toxic to Escherichia coli, which itself contains a gene, norW, encoding an NADH:(flavo)Rd oxidoreductase that has high sequence similarity to NROR (16). Nevertheless, we were able to obtain an active recombinant form of NROR (rNROR) via a fusion protein expression system. Herein we demonstrate that the complete pathway from NAD(P)H to reduction of SOR via NROR and rubredoxin can be reconstituted in an in vitro recombinant system, consistent with the hypothesis that this pathway functions in vivo, possibly in all anaerobic organisms.

MATERIALS AND METHODS

Construction of the rNROR expression vector.

All standard molecular biology techniques were performed essentially as described previously (38). The gene encoding P. furiosus NROR (PF1197) was PCR amplified using boiled genomic DNA as the template. The forward and reverse primers were 5′-CACGGTGATCATATGAAGGTAGTTATTGTTGGA-3′ (spanning −12 to +21 on the coding strand) and 5′-ATAATATACGCAGGAAGAGCCGGAGTA GAAATCTAAGAT-3′ (corresponding to +1,058 to +1,096 on the noncoding strand), respectively (Stratagene, La Jolla, Calif.). The sequences in boldface mark recognition sites for NdeI and SapI. Amplification was performed using P. furiosus DNA polymerase (Stratagene) and a Robocycler-40 thermocycler (Stratagene) with the following parameters: one cycle of denaturation at 95°C for 5 min, annealing at 50°C for 1.5 min, and extension at 72°C for 2 min. This was followed by 39 cycles with a 1-min denaturation at 95°C, 1.5-min annealing at 50°C, and 2-min extension at 72°C. The amplified 1.14-kb NROR gene was gel purified and isolated using the Gene Clean III kit (Qbiogene, Carlsbad, Calif.).

Initial attempts to clone PF1197 employed the T7 expression vector pET-21b system (Novagen, Milwaukee, Wis.), but the resulting constructs proved toxic to E. coli and could not be maintained. As a result, an intein-based fusion system was constructed by digesting the NROR gene with NdeI and SapI and ligating it into the intein-chitin binding domain (CBD) fusion vector pCYB1 (New England Biolabs, Beverly, Mass.), which was similarly digested, yielding plasmid pBVII-2. Because expression of the NROR-intein-CBD fusion is driven by the moderate-strength tac promoter in plasmid pBVII-2, it was thought that greater expression could be achieved if the NROR-intein-CBD fusion was transferred to an expression vector containing the stronger T7 promoter. As a result, the 3,427-bp NROR-intein-CBD fragment was removed from plasmid pBVII-2 using the restriction enzyme NdeI and the blunt-end cutter DraI. The NROR-intein-CBD fragment was then ligated into the T7 promoter-containing plasmid pET-21b (Novagen), restricted with NdeI and BamHI. The BamHI site was modified using the Klenow fragment of DNA polymerase (Stratagene) to give a blunt end compatible with the DraI end on the insert. The resulting plasmid pBVII-3 was then used to obtain recombinant NROR. Prior to recombinant NROR expression, the gene sequence of NROR in the pBVII-3 construct was determined in its entirety by the Molecular Genetics Instrumentation Facility (MGIF) of the University of Georgia. DNA sequences were analyzed using the computer software package MacVector (Accelrys, Burlington, Mass.).

Expression and purification of recombinant NROR.

For expression of the recombinant NROR gene in E. coli, plasmid pBVII-3 was transformed into strain BL21(λDE3), which has isopropyl-β-d-thiogalactopyranoside (IPTG)-induced expression of T7 RNA polymerase. The recombinant strain was grown in a 100-liter fermentor at 37°C using Luria-Bertani (LB) as the growth medium, supplemented with ampicillin (100 μg/ml) (University of Georgia BioXpress fermentation facility, Department of Biochemistry). Expression of the plasmid-borne NROR-intein-CBD fusion was induced by the addition of IPTG (0.4 mM) once the culture had reached an optical density at 600 nm (OD600) of 0.8.

To purify rNROR, all steps were performed under anaerobic conditions using degassed buffers. rNROR was isolated from 50 g of frozen E. coli cell paste suspended in 150 ml of buffer A (20 mM Tris-HCl [pH 8.0], 500 mM NaCl, 0.1 mM EDTA, 0.1% Triton X-100), and the cells were lysed by freeze-thaw and sonication. A cell extract was obtained by centrifugation at 100,000 × g for 60 min, and this was applied at a flow rate of 0.5 ml/min to a chitin-agarose bead column (2.5 by 10 cm; New England Biolabs) equilibrated with column buffer A. The loaded column was subsequently washed with 3 column volumes of buffer A to remove any loosely bound protein. At this point, cleavage buffer (20 mM Tris-HCl [pH 8.0], 50 mM NaCl, 0.1 mM EDTA, 0.1 mM FAD, 30 mM dithiothreitol [DTT]) was applied to the column at a flow rate of 4 ml/min until the entire column bed was equilibrated with cleavage buffer. The column was then incubated at 4°C for 24 h to allow for intein-mediated cleavage of the NROR-intein-CBD fusion and release of rNROR. Recombinant NROR protein was eluted from the column as the elution buffer (20 mM Tris-HCl [pH 8.0], 50 mM NaCl, 0.1 mM EDTA) was applied to the column at a flow rate of 2 ml/min. Fractions containing pure rNROR as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were concentrated, and the buffer was exchanged with storage buffer (50 mM Tris-HCl [pH 8.0], 2 mM dithiothreitol, 2 mM sodium dithionite, 10% glycerol) using YM-10 Centricon concentrators (Millipore, Bedford, Mass.). In an effort to show that NROR is only catalytically active when FAD is bound, rNROR was also purified as indicated above, except that FAD was not applied to the chitin-agarose column in the cleavage buffer. The absence of FAD in this rNROR preparation was confirmed by UV-visible analysis.

Enzyme assays.

NROR activity was routinely determined anaerobically by the NADPH-dependent reduction of P. furiosus rubredoxin at either 23 or 80°C. Rubredoxin reduction was measured by the decrease in absorption at 494 nm using a molar absorbance of 9.22 mM−1 cm−1 (8) for the fully oxidized protein. The reaction mixture (2.0 ml) contained 100 mM EPPS [N-(2-hydroxyethyl)-piperazine-N′-(3-propanesulfonic acid)] buffer (pH 8.0), 0.3 mM NADPH, and 20 μM rubredoxin. For assays used in the determination of kinetic parameters, NADH (0 to 0.2 mM) was also used as the electron donor and benzyl viologen (BV; 1 mM) was used as an alternative electron acceptor. Assays were also performed using native NROR for comparison (31). Recombinant rubredoxin and SOR used in these studies were obtained as indicated previously (11, 21).

rNROR reconstituted with FAD was prepared anaerobically as follows. rNROR that had been purified without FAD addition was mixed with a 10-fold molar excess of either FAD or flavin mononucleotide (FMN) and was incubated at 85°C for 15 min. Unbound cofactor was removed by gel filtration. rNROR assays were performed at 80°C using reaction mixtures (2 ml) containing 50 mM CAPS [3-(cyclohexamino)-1-propanesulfonic acid] buffer (pH 10.2), 0.3 mM NADH, and 1 mM BV. The assay was initiated by the addition of rNROR, and a molar absorbance of 7,800 M−1 cm−1 at 598 nm was used to calculate the rate of BV reduction (31). One unit of activity is equivalent to 1 μmol of BV reduced/min.

Coupled reduction of SOR and rNROR via rubredoxin was assayed using the following conditions. Recombinant P. furiosus SOR (29 μM) was placed in a sealed, anaerobic cuvette at time 0 containing degassed, 100 mM EPPS buffer (pH 8.0, 23°C) and 300 μM NADPH. Recombinant P. furiosus NROR (2.2 nM) and rubredoxin (5 μM) were then added, and the resultant reduction of SOR was monitored by the decrease in absorbance at 658 nm (extinction coefficient for SOR at 658 nm is 2,778 M−1 cm−1).

For the hydrogen peroxide assays, a standard SOD reaction mixture (2 ml) was prepared which contained 50 mM potassium phosphate buffer, pH 7.8; 0.2 mM xanthine; 3.4 μg xanthine oxidase; and 20 μM horse heart cytochrome c (22). Production of superoxide in the reaction was initiated with the addition of xanthine oxidase, and the reaction mixtures were incubated for 1 min at 23°C before further additions. Positive controls contained either 30 U of bovine liver SOD or 170 nM dithionite-reduced SOR. For the experimental samples, 170 nM each of recombinant P. furiosus SOR (21), Rd (11), and NROR that had been preincubated together anaerobically at 23°C for 2 min in 100 mM EPPS buffer (pH 8.0), 0.3 mM NADPH was added after the initial 1-min period for superoxide generation. Negative control reaction mixtures lacked either SOR, NROR, or Rd. After 3 min of incubation at 23°C, the amount of hydrogen peroxide was determined using a colorimetric method (34). For this method, 1 ml of the reaction mixture was added to 1 ml of the phenol red solution. This contained 10 mM potassium phosphate buffer, pH 7.0; 140 mM NaCl; 5.5 mM dextrose; 0.28 nM phenol red; and 8.5 U of horseradish peroxidase/ml. The resulting mixture was incubated at 23°C for 5 min. Ten microliters of 1 N NaOH was then added to increase the pH to 12.5, and the absorbance of the samples was determined at 610 nm. To quantify only the amount of hydrogen peroxide generated by enzymatic scavenging and not that produced as a result of spontaneous dismutation, control samples were prepared that contained all of the assay components but without the addition of SOD or SOR. The amount of hydrogen peroxide present in the samples was calculated using a standard curve.

Electrochemical analysis of rNROR.

The reduction potential of recombinant NROR was measured both at 23 and 80°C by cyclic voltammetry with a glassy carbon electrode. The electrochemistry cell was identical to that used by Hagen (19). Before each measurement, the electrode was polished using a 0.3 μM Al2O3 slurry and then with a 1 μM diamond spray. For analysis at 80°C, the reduction potential of rNROR (20 to 45 μM) in 50 mM MOPS (3-N-morpholinepropanesulfonic acid) buffer (pH 7.0) in a total volume of 100 μl was measured in the presence and absence of the promoter, 100 mM MgCl2. The temperature of the electrode was maintained by immersing the electrochemical apparatus in a controlled water bath. The scan rate used was 20 mV/s over the potential range of −50 to −750 mV. The working, counter, and reference electrodes were glassy carbon, Pt, and Ag/AgCl, respectively. For the 23°C measurements, rNROR (30 to 40 μM) in 50 mM MOPS, pH 7.0, was analyzed in the presence and absence of the promoters neomycin (1 mM) and MgCl2 (100 mM) with the same scan rate.

Other methods.

Protein concentrations were routinely estimated using the Bradford method with bovine serum albumin as the standard (9). The protein content of pure rNROR was also determined by the quantitative recovery of amino acids from compositional analyses performed at the Microchemical Facility at Emory University School of Medicine (Atlanta, Ga.). This analysis indicated that the protein content corresponded well (±5%) to that obtained by the Bradford method. As a result, all analytical values for the pure protein were based on the Bradford method. UV-visible spectra of oxidized and reduced forms of rNROR (33 μM in 50 mM EPPS, pH 8.0) were recorded on a Hewlett-Packard 8452A diode array spectrophotometer at 23°C. rNROR was reduced by the addition of sodium dithionite (approximately twofold molar excess over rNROR) under anaerobic conditions. The FAD content of purified rNROR was determined using the difference in absorbance of FAD at 450 nm when oxidized or reduced, using a molar absorbance coefficient of 10,300 M−1 · cm−1 at 450 nm for FAD.

Nucleotide sequence accession number

The sequence for P. furiosus NAD(P)H:rubredoxin oxidoreductase (PF1197) has been deposited in the GenBank database under accession number AE010228.

RESULTS

Expression and purification of recombinant NROR.

In order to reconstitute the proposed pathway for the reduction of superoxide by NADPH, it was necessary to recombinantly express and biochemically characterize P. furiosus NROR. The gene sequence of P. furiosus NROR was identified in the partial genomic sequence database of P. furiosus by searching with the N-terminal amino acid sequence MKVVIVGNGPGGFELAKQLSQTYEV, derived from purified native NROR (31). The corresponding gene (PF1197) consists of 1,077 bp encoding a 359-amino-acid, 39.9-kDa protein (Fig. (Fig.1).1). Analysis of the sequence revealed an FAD-binding motif (KVVIVGNGPGGFELAKQLS) and an NADH-binding domain (EAIIIGGGFIGLELAGNLA), which are consistent with the findings that native P. furiosus NROR contains one FAD molecule per monomer and that NAD(P)H is the preferential electron donor for the reduction of rubredoxin by NROR (31).

FIG. 1.
Alignment of the amino acid sequence of P. furiosus NROR with other NADH:rubredoxin oxidoreductases. The GenBank accession numbers for the NRORs other than the one sequenced in this work are the following: ...

The P. furiosus NROR gene was PCR amplified and initially cloned into the T7 expression vector pET-21b. However, no transformant containing a full-length version of the NROR gene was successfully isolated. In fact, colonies that contained plasmid with NROR insert DNA were slow to arise and were initially only pinpoint colonies. Plasmid DNA isolated from these colonies all exhibited deletions within the NROR gene (data not shown). The poor growth of colonies containing NROR coupled with the loss of NROR DNA sequence from isolated plasmids suggests that expression of the P. furiosus NROR gene in E. coli was toxic. The P. furiosus NROR gene was then cloned into the intein-CBD-fusion expression plasmid pCYB1. Recombinant NROR-intein-CBD fusion protein was successfully produced using this expression system (~91 kDa) (Fig. (Fig.2,lane2,lane 2). Presumably the production of rNROR as part of the intein-CBD fusion prevented the recombinant protein from folding into an active conformation and was therefore not toxic to the host E. coli cells. Upon cleavage of NROR from the intein-CBD fusion in the presence of a reducing agent and 0.1 mM FAD, NROR activity could be detected. The results of a typical purification of rNROR from BL21(λDE3)/pET-BVII-3 cells are summarized in Table Table1.1. Recombinant NROR could be purified after passage of E. coli cell extract through a chitin resin column. Fractions that appeared to contain only rNROR with no significant contamination were pooled and concentrated, resulting in 11.2 mg of protein from 50 g (wet weight) of cell material, determined to be pure by SDS-PAGE analysis (Fig. (Fig.2).2). This amount of recombinant protein represented 0.43% of total cellular protein. However, this is an underestimate of the actual value by approximately 10-fold, because only ~10% of the recombinant fusion protein present in the cell extract as determined by SDS-PAGE (data not shown) bound successfully to the chitin column regardless of the binding procedure used, most likely due to the masking of the chitin-binding domain by partially unfolded rNROR.

FIG. 2.
SDS-12% polyacrylamide gel electrophoresis of purified P. furiosus rNROR. Lane 1, molecular mass markers (10-kDa protein ladder; Invitrogen); lane 2, cell extract; lane 3, rNROR. The arrow marks the location of the NROR-intein-CBD fusion (~91 ...
TABLE 1.
Purification of rNROR

Biophysical properties of recombinant NROR.

Purified rNROR migrated as a 40-kDa protein as determined by SDS-PAGE analysis (Fig. (Fig.2)2) (26), which corresponds well to the calculated molecular mass of 39.9 kDa. Furthermore, the presence of FAD, as predicted by sequence analysis (Fig. (Fig.1),1), was confirmed by a UV-visible spectrum of oxidized (as purified) rNROR. The recombinant NROR exhibited peaks at 449 and 380 nm that diminish in intensity upon reduction of the protein by dithionite, which is characteristic of spectra of FAD-containing proteins (Fig. (Fig.3)3) (35). Furthermore, spectrophotometric quantitation of FAD content indicated that 54% of the purified rNROR was bound with FAD. The midpoint potential of rNROR was determined to be −173 mV at 23°C and −193 mV at 80°C (versus those of the normal hydrogen electrode [NHE]; data not shown). These values fall within the range of +31 and −320 mV, which are the reduction potential values for native P. furiosus rubredoxin (17) and NAD(P)H, respectively, that together function as the electron acceptor and donor for the electron transfer reaction catalyzed by NROR.

FIG. 3.
UV-visible spectra of oxidized (as purified) and reduced P. furiosus rNROR. The solid-line trace represents oxidized rNROR, the dotted line represents rNROR partially reduced with dithionite, and the dashed line represents rNROR fully reduced with dithionite. ...

Catalytic properties of recombinant NROR.

Because amino acid sequence analysis and the UV-visible spectrum of NROR indicated the presence of bound FAD, reconstitution experiments of apoenzyme-rNROR were performed to examine the catalytic efficiency with and without bound FAD. From NROR assays using NADH as the electron donor and benzyl viologen as the electron acceptor, it was determined that there is little activity (3.5 U/mg before heat treatment, 21.9 U/mg after heat treatment) associated with the apoenzyme (Table (Table2).2). However, when the rNROR preparation was reconstituted with a 10-fold molar excess of FAD, the activity increased to 352 U/mg. Reconstitution of rNROR with a 10-fold excess of FMN yielded inactive enzyme, indicating that catalytic activity of P. furiosus NROR requires the cofactor FAD.

TABLE 2.
Reconstitution of recombinant P. furiosus NROR

Like the native enzyme, purified rNROR was capable of catalyzing the NADH-dependent reduction of BV as well as the NADPH-dependent reduction of BV and rubredoxin (Rd). The kinetic values determined for these reactions at both 80 and 23°C are summarized in Table Table33 with comparison to kinetic values previously reported for native P. furiosus NROR (31). Like the native enzyme, rNROR exhibited a preference for catalyzing the NADPH-dependent reduction of rubredoxin over other substrates. Furthermore, when assayed at 80°C, the apparent Vmax and kcat values for the NADH-dependent reduction of BV are quite similar. Likewise, the Km of 37 μM calculated for the NADPH-dependent reduction of rubredoxin by rNROR is similar to that calculated for the native enzyme (50 μM). However, the apparent Km values for the reduction of BV by rNROR range from 4.4- to 9.6-fold higher than the corresponding native protein values. Additionally, the apparent Vmax, kcat, and kcat/Km values of 7,307 U/mg, 8,890 s−1, and 242,700 mM−1 s−1, respectively, for the NADPH-dependent reduction of Rd by rNROR represent only 36.5, 59.9, and 81.0%, respectively, of the values seen for native NROR. As with the native enzyme, the recombinant version of NROR displayed significant catalytic activity when assayed at 23°C, consistent with its proposed role in oxygen detoxification at low temperature. In this case, rNROR had a Km that was approximately 10-fold lower than that of native NROR (0.0016 compared to 0.010), and the apparent Vmax, kcat, and kcat/Km of 526 U/mg, 350 s−1, and 219,000 mM−1 s−1, respectively, are 44%, 37%, and 10-fold higher than the respective values for the native enzyme. The discrepancies observed for the kinetic parameters at 80°C can most likely be attributed to incomplete incorporation of the FAD cofactor (approximately 50%) and possibly to slight changes in the folding of the recombinant NROR enzyme relative to that of the native enzyme or perhaps less than 100% incorporation of the FAD cofactor. This slight misfolding or incomplete incorporation of cofactor likely resulted from producing the recombinant enzyme, out of necessity, as part of a fusion protein, or because the recombinant protein is expressed at temperatures considerably below the optimal growth temperature for P. furiosus (37°C as opposed to 100°C). If it is slightly misfolded, the greater flexibility of the recombinant enzyme might lead to higher catalytic efficiency at lower temperature. Although there are slight differences between the kinetic parameters calculated for the native and recombinant NRORs, both enzymes display the same overall trends in their respective values.

TABLE 3.
Kinetics of recombinant P. furiosus NROR

Reconstitution of a recombinant NADPH-dependent, superoxide reduction pathway.

It was proposed that P. furiosus NROR may participate in the reduction of SOR via reduced rubredoxin, as native NROR was shown to efficiently catalyze the reduction of rubredoxin in an NADPH-dependent reaction (31) and SOR was shown to catalyze the reduction of superoxide using reduced rubredoxin as an electron donor (22). However, the NADPH-dependent reduction of superoxide in a coupled enzyme system had not been demonstrated. With the availability of recombinant forms of all three P. furiosus proteins, NROR, rubredoxin, and SOR, the biochemical feasibility of such a coupled reaction was investigated. When SOR (29 μM) was incubated at 23°C with 300 μM NADPH, no direct reduction of SOR was observed, as illustrated in Fig. Fig.4.4. When recombinant P. furiosus NROR (2.2 nM) was then added, there was still no reduction of SOR, indicating that NROR cannot directly reduce this enzyme. However, upon addition of recombinant rubredoxin (5 μM), SOR is rapidly reduced, as shown by the steep decrease in absorbance at 658 nm. The rate of SOR reduction was 42.5 μmol/min/mg of NROR at 23°C.

FIG. 4.
Reduction of P. furiosus superoxide reductase by recombinant NROR via rubredoxin. Recombinant P. furiosus SOR (29 μM) was placed in a sealed, anaerobic cuvette at time 0 containing degassed, 100 mM EPPS buffer (pH 8.0, 23°C) and 300 μM ...

In addition, the in vitro reconstitution of an NADPH-dependent pathway for superoxide reduction was directly evaluated by analyzing for hydrogen peroxide, the hypothesized end product of SOR-catalyzed reduction of superoxide (22). As controls, superoxide was generated from xanthine/xanthine oxidase and subsequently converted to hydrogen peroxide with either bovine liver SOD or dithionite-reduced SOR. The amount of superoxide converted to hydrogen peroxide was comparable (Table (Table4).4). When SOR that had been preincubated anaerobically with NROR and Rd (170 nM each) in the presence of 0.3 mM NADPH was added to the assay mixture, the amount of hydrogen peroxide increased about twofold, which would be expected given that the SOR-catalyzed reduction of superoxide provides two equivalents of hydrogen peroxide compared to the SOD-catalyzed dismutation reaction. However, if SOR, Rd, or NROR was omitted, negligible amounts of hydrogen peroxide were detectable. Thus, these results directly demonstrate that SOR mediates reduction of superoxide to hydrogen peroxide in an NADPH-dependent manner via a coupled reaction between NROR, Rd, and SOR. Furthermore, it is important to note that unlike many reactions involving enzymes from hyperthermophilic sources, this coupled reaction proceeds rapidly at mesophilic temperatures. This is consistent with its proposed involvement in oxygen detoxification in P. furiosus during exposure to oxygen at low temperatures (22).

TABLE 4.
In vitro reconstitution of the P. furiosus NAD(P)H-dependent superoxide reduction pathway as monitored by hydrogen peroxide production

DISCUSSION

Previous studies have shown that P. furiosus superoxide reductase can catalyze the enzymatic reduction of superoxide and that rubredoxin likely provides the electrons to SOR for this reduction (22). Biochemical studies have also demonstrated that the P. furiosus enzyme NAD(P)H:rubredoxin oxidoreductase preferentially catalyzes the NADPH-dependent reduction of rubredoxin (31). Based on these findings, a model describing the pathway for the reduction of superoxide in P. furiosus was developed in which SOR reduces superoxide to hydrogen peroxide using electrons donated from Rd reduced in an NAD(P)H-dependent reduction by NROR (Fig. (Fig.5).5). Thus, this model predicts that superoxide should be enzymatically reduced in an in vitro reaction in the presence of NAD(P)H if the proteins SOR, Rd, and NROR are supplied. Recombinant versions of P. furiosus SOR and Rd had been previously biochemically characterized and were available for use in reconstituting the superoxide reduction pathway in vitro; however, a recombinant version of NROR was still required for this purpose, as only small amounts of native NROR could be readily purified directly from P. furiosus biomass. Therefore, in order to generate the in vitro NAD(P)H-dependent superoxide reduction reaction to test the predicted model, P. furiosus NROR was recombinantly produced in E. coli.

FIG. 5.
The NROR/Rd/SOR pathway for superoxide oxidation by NADPH in P. furiosus. In a reducing environment, molecular oxygen will eventually be reduced to superoxide, often by reduced transition metals, such as iron (symbolized by X→X+). Superoxide ...

The amino acid sequence of NROR (deduced from the gene sequence) contains both an NADH-binding domain and a FAD-binding domain, consistent with the reported biochemical data. Furthermore, the NROR sequence has significant similarity to the sequences of known rubredoxin oxidoreductases. NROR is 61% similar to the NADH-rubredoxin oxidoreductase from Clostridium acetobutylicum (18), 41% similar to rubredoxin reductase from Pseudomonas oleovorans, which functions in the oxidation of alkanes (13), 42% similar to E. coli NorW, a NADH:(flavo)Rd oxidoreductase implicated in nitric oxide reduction (16), and 68% similar to an NADH oxidoreductase from Thermotoga maritima (32). As expected, analysis of the rubredoxin oxidoreductase homologies reveals that there is a considerable level of homology among all of the oxidoreductases in the FAD-binding region (KVVIVGNGPGGFELAKQLS) and NADH-binding region (EAIIIGGGFIGLELAGNLA) (Fig. (Fig.1).1). Thus, both biochemical and sequence data confirm the classification of NROR as an NAD(P)H:rubredoxin oxidoreductase; however, the physiological function of the enzyme remained unknown (31).

The proposed role of rubredoxin in the recently discovered superoxide reduction pathway by SOR correlated with the known properties of NROR and indicated a possible in vivo function of P. furiosus NROR (22). Biochemical characterization of NROR indicated that it preferentially functions to reduce rubredoxin, and it has been shown that both SOR and NROR, unlike the vast majority of enzymes from hyperthermophilic sources, have considerable activity at low temperatures (23°C) (5, 6). The unusual ability of both NROR and SOR to function at low temperature reinforced their proposed interaction and provided a key observation in the development of the model of the oxygen-detoxification system in P. furiosus. This observation correlated well with the idea that exposure of P. furiosus to oxygen in its natural environment most likely occurs at low temperature, so any mechanism to remove superoxide must therefore function at low temperature (15, 20, 40). The NADPH-dependent reduction of SOR occurred only via the reduction of rubredoxin by NROR in vitro (Fig. (Fig.4).4). Moreover, this coupled reaction was shown to proceed at a rapid rate at low temperature (23°C), in keeping with the oxygen detoxification model. Furthermore, hydrogen peroxide, the hypothesized product of SOR-catalyzed reduction of superoxide, was produced only in a coupled reaction containing SOR, Rd, and NROR, whereas reaction mixtures missing any one of the three enzymes failed to produce as much hydrogen peroxide. Therefore, the direct detection of hydrogen peroxide in the coupled SOR-Rd-NROR assay provides confirmation for the superoxide reduction pathway, as outlined in Fig. Fig.55.

Homologs of SOR have been found in all anaerobic organisms so far examined (22). Having shown that P. furiosus NROR can directly participate in the SOR-mediated reduction of superoxide, it was of interest to determine whether NROR-type enzymes exist in other anaerobes. P. abyssi and P. horikoshii each contain an NROR homolog (Fig. (Fig.6),6), although both are annotated as NADH oxidases and P. horikoshii appears to lack Rd. Whether these NROR homologs have a function in the SOR pathway has yet to be demonstrated. On the other hand, the hyperthermophilic bacterium Thermotoga maritima does appear to carry out NAD(P)H- and Rd-dependent superoxide reduction, as its genome contains homologs of NROR, SOR, and Rd. This suggests that this method of oxygen detoxification, and particularly the use of NROR in this process, is not unique to archaea but most likely is a characteristic of both high-temperature (2, 22) and mesophilic anaerobes (7, 27). Interestingly, the mesophilic bacterium E. coli contains an NROR homolog, norW (42% similar to PF1197), encoding NADH:(flavo)rubredoxin oxidoreductase. It is present in a two-gene operon directly downstream of norV, encoding a flavorubredoxin (16). Biochemical analysis of NorW and NorV indicates that E. coli NADH:(flavo)rubredoxin oxidoreductase functions to reduce the rubredoxin domain present in the flavorubredoxin ultimately to reduce toxic nitric oxide (16), a role very similar to that in P. furiosus. The presence of both the NADH:(flavo)rubredoxin oxidoreductase and flavorubredoxin may have contributed to the toxicity of the unfused recombinant P. furiosus NROR in E. coli. It is possible that P. furiosus NROR interfered with the wild-type activity of these E. coli proteins, possibly depleting NAD(P)H pools and thereby compromising E. coli cellular metabolism. In support of this, a plasmid expressing a nonfusion form of P. furiosus NROR can be stably maintained in an E. coli strain that has a mutation in norV (A. Grunden, unpublished data). It is also possible that NROR may be reducing some other protein to the cell's detriment, or NROR may in fact be transferring electrons to oxygen, thereby generating toxic levels of reactive oxygen species. Additional experiments are in progress to distinguish between these various possibilities.

FIG. 6.
NROR homologs are present in various organisms. The percentage of amino acid sequence similarities compared to the sequence of P. furiosus NROR are indicated. The abbreviations are the following: Rd, rubredoxin; NorV, flavorubredoxin; NorW, NADH:(flavo)Rd ...

The reconstitution of this NROR/Rd/SOR pathway substantiates this model as a mechanism for detoxification of reactive oxygen species in P. furiosus specifically and in anaerobes in general. It further demonstrates a possible in vivo function for NROR, consistent with that of NorW in E. coli (16). With the successful production of recombinant P. furiosus NROR, kinetic analyses can be undertaken to provide more detailed information in regard to the interaction of NROR, rubredoxin, and SOR. In addition, structural studies are now feasible and could be very informative given the availability of the structures of rubredoxin (12) and SOR (43) from P. furiosus.

Acknowledgments

This research was supported by grants from the National Institutes of Health (GM 60329) and the Department of Energy (FG05-95ER20175). Support for A.G. was provided by the North Carolina Agriculture Research Station (NC02184), and support for K.M. was provided by the University of Waterloo and Natural Sciences and Engineering Research Council (NSERC, Canada).

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