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Appl Environ Microbiol. Feb 2009; 75(4): 1021–1029.
Published online Jan 5, 2009. doi:  10.1128/AEM.01425-08
PMCID: PMC2643570

O2 and Reactive Oxygen Species Detoxification Complex, Composed of O2-Responsive NADH:Rubredoxin Oxidoreductase-Flavoprotein A2-Desulfoferrodoxin Operon Enzymes, Rubperoxin, and Rubredoxin, in Clostridium acetobutylicum[down-pointing small open triangle]

Abstract

Clostridium acetobutylicum, an obligate anaerobe, grows normally under continuous-O2-flow culture conditions, where the cells consume O2 proficiently. An O2-responsive NADH:rubredoxin oxidoreductase operon composed of three genes (nror, fprA2, and dsr), encoding NROR, functionally uncharacterized flavoprotein A2 (FprA2), and the predicted superoxide reductase desulfoferrodoxin (Dsr), has been proposed to participate in defense against O2 stress. To functionally characterize these proteins, native NROR from C. acetobutylicum, recombinant NROR (rNROR), FprA2, Dsr, and rubredoxin (Rd) expressed in Escherichia coli were purified. Purified native NROR and rNROR both exhibited weak H2O2-forming NADH oxidase activity that was slightly activated by Rd. A mixture of NROR, Rd, and FprA2 functions as an efficient H2O-forming NADH oxidase with a high affinity for O2 (the Km for O2 is 2.9 ± 0.4 μM). A mixture of NROR, Rd, and Dsr functions as an NADH-dependent O2 reductase. A mixture of NROR, Rd, and rubperoxin (Rpr, a rubrerythrin homologue) functions as an inefficient H2O-forming NADH oxidase but an efficient NADH peroxidase with a low affinity for O2 and a high affinity for H2O2 (the Kms for O2 and H2O2 are 303 ± 39 μM and ≤1 μM, respectively). A gene encoding Rd is dicistronically transcribed with a gene encoding a glutaredoxin (Gd) homologue, and the expression levels of the genes encoding Gd and Rd were highly upregulated upon exposure to O2. Therefore, nror operon enzymes, together with Rpr, efficiently function to scavenge O2, O2, and H2O2 by using an O2-responsive rubredoxin as a common electron carrier protein.

The genus Clostridium, consisting of bacteria that are typical obligate anaerobes, is of great interest due to its use for bioenergy fermentation and biodegradation. The members of this genus exhibit O2-sensitive growth profiles (11, 34). The mechanism by which oxidative growth inhibition of these bacteria leads to cell death is believed to be due to a lack of enzymes such as catalase and superoxide dismutase (SOD), which can scavenge reactive oxygen species (ROS) (12, 26, 35, 37). It has been reported for one member of this species, Clostridium acetobutylicum, that although cell growth ceases during a short period of aeration, cell growth resumes once the aeration is stopped, without apparent cell damage (28). It was proposed that the observed growth cessation under aerated conditions was due to a decrease in intracellular reducing capacity caused by initiation of an NADH oxidase reaction. Subsequent to this study, we have shown that several species of Clostridium, such as C. butyricum, C. acetobutylicum, and C. aminovalericum, grow normally under O2 flow culture conditions by efficiently consuming O2 (15-18). Molecular approaches have been used to determine the enzymes required for the microaerobic growth of Clostridium species. These experiments have mainly been performed on C. acetobutylicum, an efficient acetone-butanol-fermenting bacterium whose genomic structure has been elucidated (27). In our previous study, two proteins, namely, rubperoxin and flavoprotein A1, were identified for the first time as O2-inducible proteins in C. acetobutylicum by using two-dimensional electrophoresis, and the transcripts were rapidly upregulated following exposure to O2 (17). Rubperoxin was identified as a rubrerythrin homologue and was later named rubperoxin based on its unique structure and on its functional characteristics (19). This protein was also identified as a heat-inducible protein by other investigators and was therefore named heat shock protein HSP-21 (24). These investigators reported that this protein was induced by heat, low temperature, pH, butanol, NaCl, H2O2, and O2 (10, 24). Other O2 stress-responsive genes, encoding nror operon enzymes (NROR, FprA2, Dsr, Orf2451, and flavodoxin), glutathione peroxidase homologues, bacterioferritin comigulatory protein, and thiol peroxidase, were identified in our previous study with the induction of enzyme activities for O2 and ROS scavenging (18). With the exceptions of rubperoxin and Dsr, the functions of most of these O2-inducible proteins have not been determined. Rubperoxin has been shown to function as a novel type of NAD(P)H-dependent H2O2 reductase together with an unknown proximal electron donor protein (19). The recombinant Dsr protein has been purified and is proposed to function as a superoxide reductase using spinach ferredoxin NADP+ reductase (30). However, there are still open questions concerning the identity of its proximal electron donor protein and concerning the kinetic details of the reaction.

Regarding O2-consuming activity in the Clostridium species, we have reported the purification and characterization of a H2O-forming NADH oxidase in C. aminovalericum (16). The gene encoding C. aminovalericum NADH oxidase is rapidly upregulated upon exposure to O2, and enzyme kinetic studies have revealed that this enzyme can function as an O2-consuming enzyme in vivo (the Km for O2 is calculated as 61.9 μM) (16, 18). A H2O-forming NADH oxidase homologue is not present in the C. acetobutylicum genome, though NROR does have a low homology (17% identity) to this enzyme (18). The C. acetobutylicum NROR was originally characterized as an NADH:rubredoxin oxidoreductase, although a function of NROR in protection against oxidative stress has not been elucidated (9, 29). Although we have shown that nror operon genes are responsive to O2 stress, the NROR enzyme lacks a cysteine residue at the active center, which implies that NROR lacks an NADH-dependent O2 reductase activity (18).

In this study, we investigated the function of O2-responsive nror operon enzymes by purification of the proteins and characterization of their enzymatic activity. We propose that the obligate anaerobe C. acetobutylicum possesses an efficient multienzyme complex that can scavenge O2 and ROS by using NROR as a master electron donor protein.

MATERIALS AND METHODS

Reagents.

The reagents were of the highest grade that is commercially available. Riboflavin, flavin mononucleotide (FMN), flavin adenine dinucleotide, horse heart cytochrome c, and bovine liver catalase were purchased from Sigma. Cow milk xanthine oxidase (XOD) was purchased from Roche.

Cloning and expression of NROR, Rd, FprA2, and Dsr in Escherichia coli.

The genes CAC2448 (encoding NROR), CAC2449 (encoding FprA2), CAC2450 (encoding Dsr), and CAC2778 (encoding Rd) were amplified from the initial codon to the end codon by PCR using primer pairs NROR-N (5′-ATGCATCATCATCATCATCATAAAAGCACAAAAATTTTAATC ) and NROR-C (5′-CTATAAATTATTTAATATTGC), FprA2-N (5′-ATGCATCATCATCATCATCATCCAGCTATAAAAATTAAAGAT ) and FprA2-C (5′-CTATATACTTTTGGCAAAGTCTT), Dsr-N (5′-ATGAATAACGATTTATCAAT) and Dsr-C (5′-TTATATATCTGCTTTCCATA), and Rd-N (5′-ATGAAAAAATATGTTTGTGT) and Rd-C (5′-TTATTCTTCAGATGGCTCAA), respectively, based on the genomic sequence of C. acetobutylicum (GenBank accession number NC_003030). The underlined sequences encode the six-histidine tag used for purification of rNROR and FprA2. The other proteins were overexpressed without any tag. The purified PCR products were ligated into the pET7Blue vector, resulting in the pNROR, pFprA2, pDsr, and pRd plasmids, which were then sequenced to confirm the accuracy of the nucleotide sequence and transformed into E. coli BL21 (strain Tuner DE3; Takara, Japan). The E. coli transformants were grown at 37°C in LB broth containing ampicillin (50 μg/ml) and FeSO4 (0.1 mM). When the culture reached an optical density of 0.7 (A660), the transformed gene was induced by the addition of 1 mM IPTG (isopropyl-β-d-thiogalactopyranoside) and incubation was continued for an additional 3 h. The induced cells were harvested from 10-liter cultures (20 g), were washed once with 50 mM potassium phosphate buffer (pH 7.0) containing 0.1 mM dithiothreitol (DTT), and were then resuspended in 60 ml of the same buffer. All purification procedures were carried out at 4°C or on ice. Cell-free extracts (CFEs) were obtained by disruption of the cells in a French press (140 MPa) followed by removal of the cell debris by centrifugation at 15,000 × g for 15 min. The overexpressed proteins in the CFE were then subjected to further purification as described below.

Purification of recombinant Rd.

The E. coli CFE containing recombinant Rd was fractionated by the stepwise addition of solid ammonium sulfate to give a final concentration of 46% (wt/vol). The precipitate obtained after centrifugation at 30,000 × g was dissolved in 50 mM potassium phosphate buffer (pH 5.0). This Rd-containing solution was applied to a DEAE-Sepharose fast-flow column and eluted with a linear gradient of NaCl from 100 mM to 400 mM in the same buffer. The red-colored fractions chosen for further purification were collected, combined, and applied to a gel filtration column (3.5 by 20 cm). The active fractions were concentrated with Amicon Ultra (3,000-Da cutoff; Millipore, Japan).

Purification of native NROR from microaerobically grown C. acetobutylicum.

Native NROR was purified from Clostridium acetobutylicum DSM792 (ATCC 824). Growth conditions and aeration culture were as described previously (17, 18). The C. acetobutylicum cells were grown microaerobically using a jar fermenter to achieve aeration (17). Cells (40 g, wet weight) were harvested from 16 liters of medium and were suspended in 90 ml of 50 mM potassium phosphate buffer (pH 7.0) containing 0.2 mM phenylmethylsulfonyl fluoride and 0.1 mM DTT. The cells were then disrupted by treatment with a French pressure cell at 140 MPa. Cell extracts were obtained following the removal of cell debris by centrifugation at 15,000 × g for 15 min. The supernatant (cytoplasm), obtained after ultracentrifugation (100,000 × g for 90 min), was treated with streptomycin sulfate to remove nucleic acids. After centrifugation at 30,000 × g for 15 min, the supernatant was fractionated by the stepwise addition of solid ammonium sulfate to give a final concentration of 50% (wt/vol). The precipitate obtained after centrifugation at 30,000 × g was dissolved in 50 mM potassium phosphate buffer (pH 7.0) containing 0.2 mM phenylmethylsulfonyl fluoride and 0.1 mM DTT. The enzyme solution was applied to a butyl-Toyopearl 650S column (2.5 by 37 cm) and eluted with the same buffer containing 1 M ammonium sulfate. The active fractions chosen for further purification were collected, combined, and applied to a hydroxyapatite column (3.5 by 20 cm). The column was washed with 150 mM potassium phosphate buffer (pH 7.0) and eluted with sequential application of 190 mM and 230 mM potassium phosphate buffer (pH 7.0). The eluted sample was then applied to a DEAE-Sephacel 650S column (3.5 by 20 cm). The column was washed with 10 mM Tris-HCl buffer (pH 7.0), and the enzyme was eluted with a linear gradient of 80 mM to 400 mM Tris-HCl buffer, pH 7.0. The fractions were assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the fractions chosen for further purification were combined and applied to a gel filtration column (3.5 by 20 cm). The fractions containing native NROR were concentrated with Amicon Ultra (30,000-Da cutoff; Millipore, Japan).

Purification of recombinant NROR and FprA2.

The E. coli CFE containing rNROR or recombinant FprA2 was applied to a Talon metal affinity column (Clontech, Japan) and then sequentially washed with the same buffer containing 50 mM or 100 mM imidazole. The enzyme was eluted with a buffer containing 150 mM imidazole. The fractions were assayed by SDS-PAGE, and the fractions chosen for further purification were combined and applied to a gel filtration column (3.5 by 20 cm). The fractions containing FprA2 were concentrated with Amicon Ultra (30,000-Da cutoff; Millipore, Japan).

Purification of recombinant Dsr.

The E. coli CFE in which the recombinant Dsr was overexpressed was fractionated by the stepwise addition of solid ammonium sulfate to give a final concentration of 40% (wt/vol). The precipitate obtained after centrifugation at 30,000 × g was dissolved in 50 mM potassium phosphate buffer (pH 7.0). This Dsr-containing solution was applied to a butyl-Toyopearl 650S column (2.5 by 37 cm) and eluted with a linear gradient of 1 M to 0 M ammonium sulfate dissolved in the same buffer containing FeCl3 (0.01 mM). The fractions were assayed by SDS-PAGE, and the fractions chosen for further purification were combined and applied to a gel filtration column (3.5 by 20 cm). The fractions containing Dsr were concentrated with Amicon Ultra (3,000-Da cutoff; Millipore, Japan).

Determination of the spectrum properties and protein concentration of the purified enzymes.

The purity of all of the purified enzymes was checked by SDS-PAGE and Coomassie brilliant blue staining. The identity of all of the purified proteins was confirmed by N-terminal amino acid sequencing. For each of the proteins, the sequence of the N-terminal 20 amino acids was completely identical to the sequence predicted by translation from the respective target gene. UV/visible absorption spectra of the purified proteins were recorded on a Beckman DU70 spectrophotometer using 1-cm-path-length quartz cuvettes. The molar absorption coefficient was determined for each protein by averaging the quantities of alanine, proline, valine, threonine, and tyrosine from quantitative amino acid analysis performed at Toray Research Center, Inc., using a Hitachi model L-8500 amino acid analyzer. The calculated molar extinction coefficient was determined for Rd (epsilon492 = 5,759.1 M−1 cm−1), rNROR (epsilon450 = 12,870.6 M−1 cm−1), FprA2 (epsilon450 = 14,318.7 M−1 cm−1), and Dsr (epsilon280 = 18,667.9 M−1 cm−1).

For enzymatic analyses, purified enzyme solutions were desalted in an enzyme concentrator (Amicon Ultra, 3,000-kDa to 30,000-kDa cutoff; Millipore, Japan). Briefly, the buffer in which the purified enzyme was dissolved was changed to 50 mM potassium phosphate buffer, pH 7.0, by diluting the concentrated enzyme solution 100-fold with the new buffer, followed by concentration of the enzyme to give the original volume. This dilution and concentration process was then repeated, and the final concentrated enzyme solution was subjected to an enzyme assay. The flavin content of FprA2 was determined by high-performance liquid chromatography analysis according to a previously described method (16). Flavin was identified using riboflavin, flavin adenine dinucleotide, and FMN as standards.

The specific activities shown are the averages of results from three independent measurements that varied by less than 10%.

Measurement of the NADH oxidase activity of NROR in the presence or absence of Rd.

The NAD(P)H oxidase activity of NROR, and the effect of Rd on this activity, was assayed by monitoring the decrease in dissolved-O2 concentration with an O2 electrode (YSI, OH) at 37°C. The enzymatic reaction was started by the addition of purified native or recombinant NROR, or a mixture of native or recombinant NROR and Rd, into an air-saturated 50 mM potassium phosphate buffer containing NADH (150 μM) in a reaction cuvette. One unit of NADH- and NADPH-dependent O2 reductase activity was defined as the amount of enzyme that catalyzes the reduction of 1 μmol O2 per minute. The pH optimum of this enzyme reaction was determined with 50 mM potassium phosphate buffer over a pH range of pH 5.0 to pH 8.0. The pH optima for both native and recombinant NROR:Rd O2 reductase activity were pH 7.0, and this pH was used for the experiments described below.

Measurement of the NAD(P)H-dependent Rd reductase activity of NROR.

The NAD(P)H-dependent Rd reductase activity of NROR was assayed by monitoring the reduction of Rd that was detected as a decrease in absorbance at 492 nm using a Shimadzu U-160 spectrophotometer (Shimadzu, Kyoto, Japan) and 1-cm-path-length anaerobic quartz cuvettes at 25°C. All experiments were performed under an atmosphere of O2-free argon prepared by passing 99.9999% argon through a gas-clean column (O2 trapper; Nikka Seiko Co., Tokyo, Japan). The anaerobic enzyme samples and NAD(P)H solutions were prepared in an all-glass apparatus by sequential evacuation and reequilibration with O2-free argon. Enzyme and NAD(P)H solutions were introduced into anaerobic glass cuvettes that contained anoxic reaction buffer (50 mM potassium phosphate buffer, pH 7.0) through a gastight syringe. One unit of NADH- and NADPH-dependent Rd reductase activity was defined as the amount of NROR (mg) that catalyzes the reduction of 1 μmol Rd (A492) per minute.

Measurement of the NAD(P)H-dependent O2 reductase activity of a mixture of NROR and Rd or NROR, Rd, and FprA2.

NAD(P)H-dependent O2 reductase activity was assayed by monitoring the decrease in dissolved-O2 concentration with an O2 electrode at 37°C. Reactions were started by the addition of a mixture of NROR and Rd or of NROR, Rd, and FprA2 proteins to a reaction cuvette containing 50 mM potassium phosphate buffer (pH 7.0). One unit of NADH- and NADPH-dependent O2 reductase activity was defined as the amount of enzyme that catalyzes the reduction of 1 μmol O2 per minute.

Measurement of the NADH-dependent O2 reductase activity of a mixture of NROR, Rd, and Dsr.

NADH-dependent superoxide anion reductase activity was assayed by monitoring the decrease in absorbance at 340 nm with a Shimadzu U-160 spectrophotometer at 37°C. Reactions were started by the addition of NROR, a mixture of NROR and Rd, a mixture of NROR and Dsr, or a mixture of NROR, Rd, and Dsr proteins in air-saturated 50 mM potassium phosphate buffer (pH 7.0) into a reaction cuvette. A flux of superoxide anion was then initiated by the addition of a precalibrated amount of XOD and xanthine (0.5 mM). The superoxide anion flux generated by xanthine and XOD was calibrated before and after every experiment by measuring the rate of reduction of horse heart ferricytochrome c (20 μM or 100 μM) at 550 nm (epsilon550 = 21 mM−1 cm−1) (25). One unit of NADH- and NADPH-dependent superoxide anion reductase activity was defined as the amount of NROR that catalyzes the oxidation of 1 μmol NAD(P)H per minute. The small, background NADH oxidation that was detected prior to the addition of XOD was subtracted from that measured after the addition of XOD.

Measurement of the NADH-dependent O2 and H2O2 reductase activity of a mixture of NROR, Rd, and Rpr.

Recombinant Rpr protein was purified from E. coli as described previously (19) except for the addition of FeSO4 (0.1 mM) into the E. coli culture medium, which ensured incorporation of the Fe atom into Rpr (1.8 ± 0.3 Fe/Rpr monomer). NADH-dependent O2 reductase activity was assayed by monitoring the decrease in a dissolved-O2 concentration with an O2 electrode at 37°C. One unit of NADH- and NADPH-dependent O2 reductase activity was defined as the amount of NROR that catalyzes the reduction of 1 μmol O2 per minute. NADH-dependent H2O2 reductase activity was assayed by monitoring the decrease in absorbance at 340 nm with a Shimadzu U-160 spectrophotometer using 1-cm-path-length anaerobic quartz cuvettes at 37°C. All experiments were performed under an atmosphere of O2-free argon prepared by passing 99.9999% argon through a gas-clean column (O2 trapper; Nikka Seiko Co., Tokyo, Japan). The anaerobic enzyme samples, NADH, NADPH, and H2O2 were prepared in an all-glass apparatus by sequential evacuation and reequilibration with O2-free argon. The reactions were started by the addition of a mixture of NROR and Rd or NROR, Rd, and Rpr proteins in 50 mM potassium phosphate buffer (pH 7.0) through a gastight syringe. After the addition of all of the purified enzymes into a reaction cuvette, H2O2 (0.1 mM) was added using a gastight syringe. One unit of NADH- and NADPH-dependent H2O2 reductase activity was defined as the amount of NROR that catalyzes the oxidation of 1 μmol NADH per minute.

Measurement of SOD activity.

SOD activity was assayed by monitoring the inhibition of nitroblue tetrazolium reduction (560 nm) by superoxide anion that is generated by xanthine and XOD at 37°C as described previously (2). One unit of SOD activity was defined as the amount of protein that inhibits the rate of nitroblue tetrazolium reduction by 50%.

Steady-state kinetic analyses.

Kinetic parameters of the purified enzymes were determined by the following experiments. Initial rates were determined from linear plots of product formation (or substrate disappearance). For the NADH-dependent O2 reductase assay, buffer solutions containing different concentrations of dissolved O2 were prepared by purging a N2-based gas with different O2 concentrations. The final dissolved-O2 concentration in the reaction cuvette was checked with an O2 electrode prefitted to a cuvette by a rubber seal to prevent O2 contamination from outside the cuvette. The Km value for Rd of rNROR was determined using O2 as an electron acceptor by varying the concentration of Rd with fixed concentrations of rNROR (0.01 μM), NADH (150 μM), and O2 (air saturated; 213 μM). The Km value for NADH and NADPH in the NAD(P)H-dependent O2 reductase reaction catalyzed by a mixture of rNROR and Rd was determined by varying the concentration of NADH or NADPH with fixed concentrations of rNROR (0.01 μM), Rd (2 μM), and O2 (air saturated, 213 μM). The Km value for FprA2 or Rpr in the NADH-dependent O2 reductase reaction catalyzed by a mixture of rNROR, Rd, and FprA2 or Rpr was determined by varying the concentration of FprA2 or Rpr with fixed concentrations of rNROR (0.01 μM), Rd (2 μM), NADH (150 μM), and O2 (air saturated, 213 μM). The Km value for O2 in the NADH-dependent O2 reductase reaction catalyzed by rNROR, rNROR and Rd, rNROR together with Rd and FprA2, or rNROR together with Rd and Rpr was determined by varying the concentration of O2 with fixed concentrations of rNROR (0.01 μM), Rd (2 μM), FprA2 (2 μM), Rpr (2 μM), and NADH (150 μM). The Km value for Dsr in the NADH-dependent superoxide reductase reaction catalyzed by a mixture of rNROR, Rd, and Dsr was determined by varying the concentration of Dsr with fixed concentrations of rNROR (0.01 μM), Rd (2 μM), NADH (150 μM), O2 (air saturated, 213 μM), xanthine (0.5 mM), and XOD (at a superoxide flux of 8.1 ± 0.3 μM/min). The Km value for H2O2 in the NADH-dependent H2O2 reductase reaction catalyzed by a mixture of rNROR, Rd, and Rpr was determined under anaerobic conditions by varying the concentration of H2O2 with fixed concentrations of rNROR (0.01 μM), Rd (2 μM), Rpr (2 μM), and NADH (150 μM). The Km value for Rpr in the NADH-dependent H2O2 reductase reaction catalyzed by a mixture of rNROR, Rd, and Rpr was determined by varying the concentration of Rpr with fixed concentrations of rNROR (0.01 μM), Rd (2 μM), NADH (150 μM), and H2O2 (0.1 mM). Michaelis-Menten parameters (Km and Vmax) were determined by nonlinear regression analysis using Enzyme Kinetics Module 1.3 (Sigma Plot 10.0.1; Systat Software, Chicago, IL). All values reported are the means ± standard errors for three independent experiments. The maximum turnover number, kcat, was calculated on the basis of moles of substrate oxidized or reduced per second per NROR (monomer). The errors for kcat/Km were calculated with the following formula:

equation M1

where SE1 and SE2 are the standard errors for kcat and Km, respectively.

Northern hybridization.

Northern hybridization was performed as described previously (17, 18). The RNA (15 μg) was loaded in 1.0% agarose gels and blotted onto nylon membranes (Hybond N+; Amersham, Japan). The membranes were probed with the entire coding sequence for glutaredoxin (Gd gene; GenBank accession no. CAC2777) or rubredoxin (Rd gene; GenBank accession no. CAC2778). The Gd and Rd genes were amplified by PCR using chromosomal DNA from C. acetobutylicum as a template and the following oligonucleotide primer pairs: 5′-ATGGTAAAAGTATATTCAAC-3′ and 5′-TTATTTTAAATTTAAAAGTT-3′ (Gd gene) and 5′-ATGAAAAAATATGTTTGTGT-3′ and 5′-TTATTCTTCAGATGGCTCAA-3′ (Rd gene).

RESULTS

Purification of recombinant rubredoxin from E. coli and native NADH:rubredoxin oxidoreductase from C. acetobutylicum.

In this study, in order to characterize the function of O2-inducible NROR in oxidative stress protection, native NROR was purified. We first purified recombinant C. acetobutylicum rubredoxin (Rd gene; GenBank accession no. CAC2778) by using an E. coli recombinant expression system. The purified Rd protein exhibited a red color, as two major peaks at 380 nm and 492 nm (see Fig. S1B in the supplemental material). This observed spectrum, consisting of two major peaks originating from the Fe-S cluster of Rd, is in good agreement with previously published values for rubredoxin from Clostridium pasteurianum (22). The purity of purified Rd was assessed by gel electrophoresis and Coomassie blue staining (see Fig. S1A in the supplemental material). The molar absorption coefficient of purified Rd was determined by quantitative amino acid analysis.

Using Rd as an electron acceptor in NADH-dependent rubredoxin reductase assays, we then purified the native NADH:rubredoxin oxidoreductase from microaerobically grown C. acetobutylicum cell extracts. After the first step of column chromatography, NROR activity eluted together with an NADH oxidase activity as a major single peak. This NADH oxidase activity was significantly decreased after passage through a hydroxyapatite column, which was the second column chromatographic purification step. High NADH oxidase activity was restored to the NROR fractions by the addition of purified Rd and FprA2. These data suggested that the observed decrease in NADH oxidase activity was due to the chromatographic separation of these associated proteins. Electrophoresis of the purified NROR on an SDS-polyacrylamide gel followed by Coomassie brilliant blue staining yielded a single band of approximately 43 kDa (see Fig. S1A in the supplemental material). The N-terminal amino acid sequence of the purified enzyme was determined, and the sequence of the N-terminal 20 amino acids was completely identical to that for the previously characterized C. acetobutylicum NROR (GenBank accession no. AAK08126). The purified NROR was yellow and showed two absorption peaks at 380 nm and 452 nm (see Fig. S1B in the supplemental material). NROR has previously been reported to function as an NADH-dependent Rd reductase in vitro (29). However, the in vivo function of this enzyme has not yet been elucidated (9, 29). It has also been reported that reactivity of NROR toward O2 could not be detected (29). In this study, purified NROR exhibited little NADH- and NADPH-dependent O2 reductase activity. The specific activity of NROR in an NAD(P)H oxidase reaction in air-saturated buffer was 4.2 U/mg protein or 4.4 U/mg protein when 150 μM of NADH or NADPH, respectively, was used as an electron donor. In each case, the pH optimum of the reaction was pH 7.0. The final product of the NADH- and NADPH-dependent oxidase reaction was H2O2, which was produced stoichiometrically by two reducing equivalents from O2 (data not shown).

Purification and characterization of recombinant NROR in E. coli.

To further characterize the function and kinetic properties of the NROR protein, a large amount of protein was required. For this purpose, rNROR was expressed in E. coli and purified. The purified rNROR showed almost the same characteristics as those of the native enzyme, including spectral analysis results (see Fig. S1B in the supplemental material), specific activity of NAD(P)H oxidase, and reaction properties. The purified rNROR used NADH and NADPH as electron donors. The specific activity of rNROR in the NAD(P)H oxidase assay in air-saturated buffer was 7.1 U/mg protein or 7.4 U/mg protein when 150 μM of NADH or NADPH, respectively, was used as an electron donor. The NADH- and NADPH-dependent O2 reductase reaction catalyzed by rNROR also stoichiometrically reduced O2 to H2O2 (Fig. (Fig.1),1), and the pH optimum of this reaction was pH 7.0.

FIG. 1.
NADH-dependent O2-consuming activity of mixtures of rNROR, Rd, Rpr, and FprA2 and determination of the final product of the O2 reduction. Air-saturated 50 mM potassium phosphate buffer, pH 7.0, containing 300 μM NADH, was introduced into a reaction ...

Unless otherwise indicated, the following enzymatic characterization of NROR was performed using rNROR. Under anoxic conditions, rNROR (0.7 nM) catalyzed a hyperreducing activity of Rd (70 μM), calculated as 1,940 U/mg rNROR (kcat = 1.3 × 103 [s−1]) when NADH (150 μM) was used as an electron donor and 408 U/mg rNROR (kcat = 288 [s−1]) when NADPH (150 μM) was used as an electron donor at pH 7.0. Under aerobic conditions, rNROR catalyzed both NADH- and NADPH-dependent O2 reductase activity, but the affinity for NADPH was very low compared to that for NADH (the Kms for NADH and NADPH were 0.7 ± 0.1 μM and 124.3 ± 9.1 μM, respectively, when O2 [air-saturated buffer in which the dissolved-O2 concentration is 213 μM at 37°C] was used as an electron acceptor). The following enzymatic assay and kinetic studies were performed using NADH as an electron donor. The Km of rNROR for O2 and the Vmax of the NADH oxidase reaction are listed in Table Table1.1. The affinity of this reaction for O2 was increased by the addition of Rd. The Km for Rd of rNROR was 0.54 ± 0.1 μM when O2 (air-saturated buffer in which the dissolved-O2 concentration is 213 μM at 37°C) was used as an electron acceptor. Investigation of the enzymatic reaction of rNROR with other associated proteins will be described in a later section.

TABLE 1.
Steady-state kinetic parameters for the NADH-dependent O2 reductase activities

Purification of recombinant FprA2 and Dsr.

As described in our previous study, nror operon genes are transcribed tricistronically with fprA2 and dsr (18). In addition, the dsr gene can also be regulated independently of the nror promoter (18). The FprA2 protein has a homology with FprA homologues found in Desulfovibrio gigas (GenBank accession no. AAG34792) (7) and in Moorella thermoacetica (GenBank accession no. Q9FDN7) (5) (28.3% and 28.5% identity, respectively). These proteins have been shown to function as Rd-dependent oxidases or nitric oxide reductases. Dsr is homologous (36% identity) to the Dsr protein of Treponema pallidum (GenBank accession no. AAC65791), which functions as a superoxide reductase (14, 21). To characterize the function of FprA2 and Dsr, these proteins were overexpressed in E. coli and purified. The purity of the recombinant enzymes was determined by SDS-PAGE. Following electrophoresis, staining of the SDS-polyacrylamide gel with Coomassie brilliant blue yielded single bands of approximately 43 kDa and 13 kDa for purified FprA2 and Dsr, respectively (see Fig. S1A in the supplemental material). N-terminal amino acid sequencing of each of these purified enzymes revealed a perfect match to the sequence predicted by translation from the respective target gene.

The spectrum of FprA2 was dominated by the flavin moiety showing two major peaks at 377 nm and 450 nm (see Fig. S1B in the supplemental material). Gel filtration of the purified FprA2 protein yielded a single peak whose elution volume corresponded to an estimated molecular mass of 180 kDa. These data demonstrate that the purified FprA2 protein is a homotetramer. Flavin analyses confirmed a cofactor content of 0.9 ± 0.2 FMN per FprA2 monomer (data are the means ± standard deviations of results from three independent analyses).

Purified C. acetobutylicum Dsr showed no significant absorption spectrum except at 280 nm (see Fig. S1B in the supplemental material). When the purified protein was treated with K3[Fe(CN)6] as an oxidant, the obtained spectrum showed an increased absorption centered at 631 nm (see Fig. S1B, inset, in the supplemental material), which is attributed to the ferric form of the iron center (21). These results indicated that the Dsr protein was purified in its reduced form. The spectra that we obtained for Dsr are in good agreement with those reported for Dsr of T. pallidum (21).

Characterization of the reaction of rNROR with Rd and FprA2.

To characterize the function of nror operon enzymes, we analyzed the enzymatic properties of a mixture of purified recombinant proteins of NROR, Rd, FprA2, Dsr, and Rpr. When FprA2 was added to a reaction mixture containing rNROR and Rd, the NADH oxidase activity was significantly enhanced. The Km for FprA2 in this reaction was 0.12 ± 0.01 μM in the presence of 0.01 μM rNROR and 2 μM Rd. In the absence of FprA2, the reaction product of the NADH-dependent O2 reduction catalyzed by rNROR or rNROR mixed with Rd was H2O2 (Fig. (Fig.1).1). This reaction product changed to H2O when FprA2 was added to the mixture (Fig. (Fig.1).1). The small amount of H2O2 detected corresponds to the amount of H2O2 produced from the reaction of rNROR and Rd. Therefore, the NADH-dependent O2 reductase activity catalyzed by a mixture of rNROR, Rd, and FprA2 reduces O2 by four reducing equivalents to H2O, as summarized in the following equation: 2NADH + 2H+ + O2 = 2NAD+ + 2H2O.

A steady-state kinetic analysis of this reaction was performed, and the Km for O2 was 2.9 ± 0.4 μM (Table (Table1;1; also see Fig. S2 in the supplemental material). This level of O2 corresponds to the concentration of saturated, dissolved O2 that is present in an atmosphere containing 0.3% O2 at 37°C. The Vmax value was calculated at 62.7 ± 0.8 U/mg rNROR. Taking into account the final product of O2 reduction, as well as the kinetic properties of the reaction, such as kcat/Km, these data indicate that the NROR-Rd-FprA2 system must be a key complex of O2 scavenging.

Characterization of the reaction of rNROR with Rd and Dsr.

We have previously proposed that the function of Dsr is that of a superoxide reductase based on its structural similarity to the Dsr of Treponema pallidum. In a recent report, recombinant C. acetobutylicum Dsr (Strep tagged for purification) was suggested to function as an NADPH-dependent superoxide reductase using Spinach ferredoxin NADP+ reductase (30). In the present study, the native-form Dsr protein was newly characterized with regard to its enzymatic and spectral properties and its enzymatic kinetics in the presence of its proximal electron donor protein. The spectral properties of Dsr are described in earlier paragraphs. The purified Dsr protein exhibited low SOD activity (83.7 ± 6.1 U/mg protein) in the absence of an electron donor. An SOD activity has been reported for the Dsr protein from T. pallidum (35 U/mg protein) (21), P. furiosus (200 U/mg protein) (13), and a Desulfovibrio species (20 to 70 U/mg protein) (20). To determine the function of Dsr as an NROR- and Rd-dependent superoxide anion reductase, the effect of Dsr on NADH oxidation was analyzed in the presence of xanthine and XOD as the superoxide anion generator (Fig. (Fig.2;2; also see Fig. S3 in the supplemental material). The reaction of xanthine and XOD generates O2 and H2O2 from the reduction of O2 (6). The superoxide anion flux was determined as equal to the rate of reduction of cytochrome c (1, 25). The calculated rate of the superoxide anion flux generated by 0.5 mM xanthine and 1 μl of purchased XOD added to 1 ml of a reaction mixture was 8.1 ± 0.3 μM/min. In the presence of rNROR and Rd, Dsr activated NADH oxidation in the presence of xanthine and XOD. In the absence of Rd or Dsr, NADH oxidation was not activated. The NADH-dependent O2 reductase activity, catalyzed by the mixture of rNROR and Rd, was not influenced by the addition of 0.1 μM to 10 μM Dsr (data not shown). When various amounts of H2O2 were added to the reaction mixture (5 μM, 20 μM, 50 μM, and 100 μM), no increase of NADH oxidation was observed (Fig. (Fig.2).2). These results indicated that the substrate of Dsr is superoxide anion. The specific activities of the NADH-dependent O2 reductase activity catalyzed by the mixture of rNROR, Rd, and Dsr were 17.9 ± 0.13 U/mg rNROR and 27.3 ± 0.6 U/mg rNROR at superoxide anion fluxes of 8.1 ± 0.3 μM/min and 16.4 ± 0.5 μM/min, respectively. Kinetic parameters of the NADH-dependent O2 reductase reaction are shown in Table Table22.

FIG. 2.
Detection of NADH-dependent O2 reductase activity catalyzed by a mixture of rNROR, Rd, and Dsr proteins. Air-saturated 50 mM potassium phosphate buffer, pH 7.0, containing 150 μM of NADH, was introduced into a reaction cuvette at 37°C. ...
TABLE 2.
Steady-state kinetic parameters of the enzymatic reactions that scavenge ROS in the rNROR-Rd protein mixturea

Characterization of the reaction of rNROR with Rd and Rpr.

Rubperoxin, a reverse-type rubrerythrin homologue, was previously determined to function as an efficient scavenger of H2O2, preferentially using NADH together with an unknown proximal electron donor protein (19). We speculated that NROR might be an electron donor protein for this reaction since rubperoxin has a conserved rubredoxin motif in its N terminus (17, 19). In the presence of O2, the mixture of rNROR, Rd, and Rpr proteins catalyzes NADH-dependent H2O-forming oxidase activity (Fig. (Fig.2).2). The Km for O2 of this reaction (303 ± 39 μM) is higher than the dissolved-O2 concentration in air-saturated medium (approximately 210 μM at 37°C). The affinity of this reaction for O2 was 100-fold lower than that of the mixture of rNROR, Rd, and FprA2. Furthermore, the kcat/Km value indicated that the oxidase activity of this reaction was significantly inferior to the oxidase activity generated by the mixture of rNROR, Rd, and FprA2 and to that of the Clostridium aminovalericum H2O-forming NADH oxidase (Table (Table2)2) (16). Purified rNROR exhibited NADH-dependent H2O2 reductase activity in the presence of Rd and Rpr (Fig. (Fig.3).3). This reaction showed a high affinity for H2O2, but this affinity for H2O2 was too high for the Km to be accurately measured. This is because NADH oxidation after the addition of H2O2 follows zero order kinetics down to the detection limit of NADH (2 μM) at 340 nm. Therefore, the Km for H2O2 was estimated to be less than 1 μM (Table (Table2;2; also see Fig. S4 in the supplemental material). The NADH-dependent H2O2 reductase activity increased as the concentration of Rpr increased, whereas the NADH-dependent O2 reductase activity did not (Table (Table3).3). The Km for Rpr in the NADH-dependent rNROR:Rd:Rpr O2 reductase reaction was 0.19 ± 0.01 μM in air-saturated buffer, and the Km for Rpr in the NADH-dependent rNROR:Rd:Rpr H2O2 reductase reaction was 35.9 ± 5.6 μM under the saturated H2O2 condition (0.1 mM). The Vmax of the NADH-dependent H2O2 reductase reaction was estimated to be 1,089 ± 123 U/mg rNROR, and the kcat/Km was 20.9 ± 4.0 μM−1 s−1 (Table (Table2).2). These results indicated that the rNROR-Rd-Rpr system functions as an efficient NADH-dependent H2O2 scavenging reaction and might be involved in O2 scavenging reactions under highly aerated conditions, such as air-saturated conditions. A protein mixture of rNROR, Rd, FprA2, and Rpr catalyzed a H2O-forming NADH oxidase activity in which no H2O2 was detected (Fig. (Fig.1).1). This result indicated that rNROR could transfer electrons to both FprA2 and Rpr via a common electron carrier protein, Rd (see Fig. Fig.55).

FIG. 3.
NADH-dependent H2O2 reductase activity of mixtures of rNROR, Rd, and Rpr. Assays were performed under anoxic conditions (see Materials and Methods). The indicated protein combinations were added into the reaction cuvette at the time indicated by the arrow. ...
FIG. 5.
Scheme of the NADH-dependent O2, O2, and H2O2 detoxification complex composed of NADH:rubredoxin oxidoreductase (NROR), rubredoxin (Rd), flavoprotein A2 (FprA2), desulfoferrodoxin (Dsr), and rubperoxin (Rpr) in the obligate anaerobe C. acetobutylicum ...
TABLE 3.
Specific activities of O2 and ROS-scavenging enzyme reaction for native NROR and recombinant NROR

Finally, native NROR purified from C. acetobutylicum was assayed to determine the interaction with other proteins in assays similar to those described for rNROR, which were designed based on the kinetic parameter obtained for rNROR. The specific activities for native NROR with nror operon enzymes and Rpr were similar to those for rNROR (Table (Table33).

A glutaredoxin-rubredoxin operon is O2 responsive.

The C. acetobutylicum genome carries a single rubredoxin gene (Rd gene; GenBank accession number CAC2778). The Gd gene (GenBank accession number CAC2777), which encodes a glutaredoxin homologue, is located 61 bp upstream of the 5′ region of the Rd gene and is transcribed in the same direction as the Rd gene (Fig. (Fig.4).4). To determine if this Gd-Rd operon might play a role in the response to oxidative stress, a Northern blot analysis of Gd-Rd mRNA expression in the presence or absence of oxidative stress was performed using total RNA extracted from C. acetobutylicum. The results show that the Gd-Rd gene is dicistronically transcribed and strongly upregulated following 10 min of microoxic aeration. Therefore, not only rubredoxin but also glutaredoxin must be involved in oxidative stress response in C. acetobutylicum.

FIG. 4.
Response of an operon encoding a rubredoxin and glutaredoxin homologue to oxidative stress. (A) Schematic diagram of an operon of the Gd gene (gd; CAC2777) and the Rd gene (rd; CAC2778), encoding a glutaredoxin homologue and rubredoxin, respectively. ...

DISCUSSION

In this study, we aimed to determine the function of the O2-responsive proteins in C. acetobutylicum. The enzyme NAD(P)H:rubredoxin oxidoreductase has been proposed to have a central role in the protection of anaerobic bacteria, such as sulfur reducers and a hyperthermophile. In Desulfovibrio gigas, NROR is composed of two subunits, with molecular masses of 27 kDa and 32 kDa (4). D. gigas NROR specifically binds NADH but not NADPH and has been shown to possess hyperreactivity of Rd reduction. Rd has been shown to function as an electron carrier protein in the reduction of rubredoxin:oxygen oxidoreductase (Roo), and the serial interaction of NROR, Rd, and Roo has been proposed as a central reaction in the reduction of O2 to H2O (7, 36). In P. furiosus, a hyperthermophilic archaeon, the NROR enzyme, exists as a monomer with a molecular mass of 45 kDa (8, 23). The P. furiosus NROR is homologous with C. acetobutylicum NROR (29% identity) but differs from the latter in that it is more specific to NADPH (8, 23). Purified recombinant P. furiosus NROR was shown to catalyze an Rd-dependent superoxide reductase reaction together with a recombinant P. furiosus superoxide reductase (8). The existence of an NROR-dependent O2 detoxification system is unclear in P. furiosus.

In this study, both native and recombinant C. acetobutylicum NROR proteins were purified. Both proteins exhibited NADH- and NADPH-dependent H2O2-forming oxidase activity. However, their affinity for NADPH was significantly lower than that for NADH. The addition of Rd weakly activated the H2O2-forming NADH oxidase reaction but did not significantly increase the affinity for O2. The further addition of FprA2 significantly enhanced its affinity to O2, with a high turnover number, and furthermore, the reaction was converted from a H2O2-forming to a H2O-forming reaction. FprA2 homologues in Moorella thermoacetica and Desulfovibrio species have been described, and the kinetic parameters of these FprA homologues with regard to oxygen metabolism have been investigated (32, 33). FprA of M. thermoacetica shows an NADH:FprA O2 reductase activity when coupled with Hrb (high-molecular-weight rubredoxin that contains a rubredoxin motif) as an electron donor protein (32). FprA had a higher affinity for NO, with an apparent Km of 4 μM, than for O2, for which the apparent Km was 26 μM. The activity of FprA from D. vulgaris was also investigated using the Hrb protein from M. thermoacetica as an electron donor protein, and this FprA protein also had a higher Km for NO (the apparent Km was 19 μM) than for O2 (the apparent Km was 24 μM) (33). The conclusion from these studies was that these two FprA homologues are involved in NADH-dependent NO reduction. The FprA homologue from methanogenic bacteria, F420H2 oxidase, was also shown to have a high affinity for O2 (the apparent Km was 2 μM) in an assay that measured the oxidation of protein-bound flavins by O2 (31). It was proposed that F420H2 oxidase catalyzes O2 reduction to H2O by coupling with Frh (F420-reducing hydrogenase) and Mtd (F420-dependent methylenetetrahydromethanopterin dehydrogenase) as proximal electron donor proteins (31). In the present study, the C. acetobutylicum NROR-Rd-FprA2 system was estimated to be enough to scavenge a trace of O2 under hypoxic conditions. We have shown that rubperoxin can also catalyze the H2O-forming NADH oxidase reaction in the presence of NROR and Rd. However, the kinetic parameters of the NROR-Rd-Rpr reactions indicated that the NROR-Rd-Rpr system is able to reduce O2 under highly aerated conditions but is not an efficient O2 scavenger under hypoxic conditions. Due to the high affinity of the NROR-Rd-Rpr system for H2O2, and its extremely high Vmax and kcat/Km values (Table (Table2),2), we propose that the main function of Rpr in vivo is that of an NADH-dependent H2O2 scavenger.

Rd was originally identified as an electron carrier protein with an unknown function and is widely distributed among anaerobes. Recently, Rd was shown to be involved in superoxide reduction, and a rubredoxin:oxygen oxidoreductase activity was described to occur in a variety of anaerobes (1, 3, 4, 13). To our knowledge, little is known concerning the transcriptional response of rubredoxin to oxygen. In this study, we have shown that a gene encoding rubredoxin is strongly, and rapidly, upregulated in response to oxygen, indicating that the role of rubredoxin in Clostridium is in protection against oxidative stress. The role of the small redox protein (glutaredoxin-like protein) that is cotranscribed with Rd is still unclear.

In summary, we propose that Clostridium acetobutylicum possesses the NADH-dependent O2, O2, and H2O2 detoxification complex shown in Fig. Fig.5.5. Many O2-inducible proteins have been identified in C. acetobutylicum, such as A-type flavoprotein A1, bacterioferritin comigulatory protein, thiol peroxidase, glutathione peroxidase-like proteins, flavodoxin, and a small redox protein, glutaredoxin (18). While these proteins remain to be characterized in detail, it seems very likely that these proteins play a role in cellular defense against oxidative stress.

Supplementary Material

[Supplemental material]

Acknowledgments

This work was supported by a grant-in-aid for Scientific Research from the Japan Society for the Promotion of Science (to S.K.)

We thank Tohru Kodama and Junichi Nakagawa for valuable discussions. We also thank Yusuke Watamura, Masaki Ono, Yusuke Tanimori, and Tomoya Maruo for helpful technical assistance at Tokyo University of Agriculture.

Footnotes

[down-pointing small open triangle]Published ahead of print on 5 January 2009.

Supplemental material for this article may be found at http://aem.asm.org/.

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