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FEBS Lett. Author manuscript; available in PMC Jan 5, 2010.
Published in final edited form as:
FEBS Lett. Jan 5, 2009; 583(1): 241–245.
Published online Dec 11, 2008. doi:  10.1016/j.febslet.2008.12.004
PMCID: PMC2629652

Reductive dioxygen scavenging by flavo-diiron proteins of Clostridium acetobutylicum


Two flavo-diiron proteins (FDPs), FprA1 and FprA2, are up-regulated when the strictly anaerobic solvent producer, Clostridium acetobutylicum, is exposed to dioxygen. These two FDPs were purified following heterologous overexpression in E. coli as N-terminal Strep-tag fusion proteins. The recombinant FprA1 and FprA2 were found to be homodimeric and homotetrameric, respectively, and both FDPs functioned as terminal components of NADH oxidases (NADH:O2 oxidoreductases) when using C. acetobutylicum NADH:rubredoxin oxidoreductase (NROR) and rubredoxin (Rd) as electron transport intermediaries. Both FDPs catalyzed the four-electron reduction of molecular oxygen to water with similar specific activities. The results are consistent with these FDPs functioning as efficient scavengers of intracellular dioxygen under aerobic or microoxic growth conditions.

Keywords: Clostridium acetobutylicum, O2 reduction, flavo-diiron, rubredoxin

1. Introduction

Obligately anaerobic microorganisms by definition cannot use molecular oxygen as terminal electron acceptor for growth. Obligate anaerobes are, nevertheless, far from defenceless against periodic influxes of O2 and its even more reactive reduced forms, superoxide and hydrogen peroxide. While aerobic organisms use catalase or superoxide dismutases to lower high levels of hydrogen peroxide (H2O2) or superoxide (O2), anaerobes use their relatively high levels of reducing equivalents for reduction rather than dismutation of these molecules, thereby avoiding the regeneration of O2 [1,2]. Enzymes catalyzing reductive superoxide and peroxide scavenging, namely, superoxide reductase (SOR), and rubrerythrin (Rbr), respectively, have been characterized from a number of anaerobic bacteria and archaea [37].

Many anaerobic bacteria also share the ability to reductively scavenge molecular oxygen directly, thereby rapidly re-establishing anaerobiosis upon intermittent exposure to a dioxygenic environment and protecting crucial O2-sensitive metabolic enzymes [1,2]. These scavenging enzymes typically function as NADH:O2 oxidoreductases (NADH oxidases) and can be distinguished by their production of either hydrogen peroxide or water from dioxygen reduction [810]. The strictly anaerobic, gram-positive solvent producer, Clostridium acetobutylicum, can withstand air exposure upon activation of its oxidative stress response, resulting in rapid cellular O2 consumption and high NADH oxidase activity in crude extracts [11]. However a corresponding gene or protein has not been conclusively identified in this organism up to now. Upon exposure of C. acetobutylicum to aerobic or microoxic (5% O2) atmospheres, or during activation of its PerR (peroxide response regulator) regulon only a very few proteins were detectably up regulated, most prominently Hsp21, which was subsequently identified as reverse rubrerythrin (revRbr) [12,13]. Although revRbr may participate in O2 scavenging, we have shown that its preferred substrate is H2O2 [7].

Two other candidates for O2-scavenging enzymes in C. acetobutylicum are the putative flavo-diiron proteins (FDPs), FprA1 and FprA2. Expressions of their respective genes, cac1027 and cac2449, are inducible by exposure to a microoxic atmosphere [13,14] and both of these FDPs were shown to be highly abundant in an aerotolerant perR deletion strain of C. acetobutylicum [12, unpublished microarray data]. Cac2449 was identified as part of an O2-responsive gene cluster [14], which also encodes an NADH:rubredoxin oxidoreductase (NROR) (cac2448) [7], and a SOR (cac2450) [6]. FDPs are widespread in anaerobic bacteria and archaea [1517], and even occur in a few primitive eukaryotes [18]. The characteristic features of FDPs are an N-terminal non-heme diiron domain and a C-terminal flavodoxin-like domain [1518]. The amino acid sequence motifs for both of these domains, as well as all the iron-ligating residues, are conserved in C. acetobutylicum FprA1 and FprA2 (see Fig. 1 and Table S1). FDPs from several sources have been shown in vitro to function as the terminal components of NADH oxidases [1518]. This activity requires the participation of electron transfer intermediary proteins, such as NROR and rubredoxin (Rd), between NADH and FDP. Genes encoding the small electron transfer protein, Rd or Rd-like protein, are in fact, co-transcribed with those of FDP homologues in other bacteria [17,19,20]. The NADH oxidase activity has led to the proposal that at least some FDPs function as O2 scavengers in vivo. To our knowledge only putative FDPs from clostridia have been shown to be up-regulated upon O2 exposure [14,21], yet no clostridial FDPs have been characterized. We, therefore, investigated whether the cac1027 and cac2449 gene products are in fact FDPs, whether they can function as the terminal components of NADH oxidases and whether this activity is consistent with an O2 scavenging function in vivo.

Fig. 1
Amino acid sequence alignment of the N-terminal diiron domains of FDPs from Moorella thermoacetica (Mta), D. gigas (Dgi), (a.k.a. rubredoxin:oxygen oxidoreductase, Roo), and C. acetobutylicum FprA1 and FprA2. Identities among the four proteins are shaded ...

2. Materials and Methods

2.1 Reagents, enzymes and standard procedures

Reagents and buffers used for cloning, purification and enzyme assays were at least of analytical grade. Restriction endonucleases and DNA ligase, obtained from NEB and Pwo polymerase from Peqlab were either used according to the manufacturer’s description or as described elsewhere [22]. Unless indicated otherwise, chemicals were purchased either from either Sigma Aldrich or Applichem and were at least of analytical grade. Amino acid sequence alignments were carried out using the Clustal W software [23]. Iron and protein analyses were carried out as previously described [17].

2.2 Cloning, expression, and purification of proteins

The genes encoding FprA1 (cac1027) and FprA2 (cac2449) were amplified from chromosomal DNA of C. acetobutylicum by PCR using the oligonucleotides P1_CAC1027-BamHI 5′-AAAAGGATCCAGTGCTGAAAAGCTTTGTG-3′ P2_CAC1027-PstI 5′-AAAACTGCAGTAATAAACCTATAAAATCATC-3′ for fprA1, and P1_CAC2449-BamH1 5′-AAAAGGATCCCCAGCTATAAAAATTAAAG-3′ and P2_cac2449-PstI 5′-AAAACTGCAGTATACTTTTGGCAAAGTC-3′ for fprA2 as primers, introducing BamHI and PstI restriction sites. The resulting fragments were ligated in the pASK-IBA7+ expression vector (IBA-Go) which allows expression of strep tag fusion proteins from an anhydrotetracyclin (AHT) inducible promoter [24]. The resulting plasmids pI7fprA1 and pI7fprA2 were transformed in competent E. coliDH5α cells. The recombinant strains were grown in 500 ml of Luria-Bertani (LB) medium with 100 μg ml−1 of ampicillin at 30°C and 120 rpm on a rotary shaker. At an OD600 of 0.3–0.5, 0.2 μg ml-1 of AHT were added to induce protein expression. Cells were harvested by centrifugation following growth for 10–12 hours. Strains expressing recombinant Rd and NROR from pI3rbocac and pTnrorcac, respectively, were grown as described previously [6,7]. Cell pellets were either stored for up to 7 days at −20°C or immediately used as a source of cellular protein. All further purification steps of the heterologous proteins used in this study (Rdcac, NRORcac, FprA1 and FprA2) were carried out as described previously [7]. The protein content in the elution fractions was determined using the Bradford assay [25]. Reduced forms of the proteins were obtained by the addition of a stoichiometric amount of sodium dithionite. Purification of the proteins was analyzed using 12.5% Coomassie-stained SDS–PAGE gels.

A more thermostable NROR from Thermotoga maritima (NRORtma) was used for determination of Michaelis-Menten parameters. (The T. maritima NROR gene (TM0754) is adjacent to that encoding the T. maritima FDP homolog (TM0755)). E. coli expression strains for N-terminal His-tagged NRORtma (TM0754) and Rd (TM0659) from T. maritima were obtained from the Joint Center for Structural Genomics (http://www.jcsg.org/) and are described on the Harvard Institute of Proteomics Plasmid ID website (http://plasmid.med.harvard.edu/PLASMID/GetCloneDetail.do?cloneid=85043&species=). The expression strains were grown in LB at 37 °C to an OD600 of 0.6–1.0, and protein expression was induced with 0.02 % arabinose. The cultures were incubated for an additional 3–4 hours at 37 °C, and the cells were then harvested and lysed by sonication. The recombinant NRORtma and Rdtma were purified from the cell lysate using Talon® columns (Clontech) with an elution buffer consisting of 50 mM MOPS, 250 mM NaCl, 250 mM imidazole, pH 7.3.

2.3 Determination of molecular weights

Molecular weights of the native proteins were determined by analytical gel filtration and fast pressure liquid chromatography (FPLC, Pharmacia Biotech) using a Superose 12 10/300 column (GE Healthcare) and 50 mM Tris-HCl pH 8.0, 0.1 mM EDTA, and 150 mM NaCl as the mobile phase. The column was calibrated with proteins of known sizes: aldolase, 158 kDa; albumin, 67 kDa; ovalbumin, 43 kDa; and chymotrypsinogen, 25 kDa.

2.4 NADH oxidase and NADH:nitric oxide oxidoreductase activities

NADH-dependent oxidase activities were measured spectrophotometrically essentially as previously described [6,7] at 25 °C in 0.4 ml of air-saturated 50 mM N-morpholinopropanesulfonic acid (MOPS) buffer + 0.1 mM EDTA pH 7.0. The standard assay contained in the order of addition 100 μM of NADH, 2 μM of Rdcac, 0.1 μM of NRORcac, and 1 μM (monomer concentrations) of either FprA1 or FprA2. Protein concentrations were determined by the Bradford method [25] and the molecular weights. The rate of decrease in absorbance at 340 nm resulting from NADH oxidation was monitored using a Ultrospec 3000 spectrophotometer (Pharmacia Biotech). One unit of activity was defined as the amount of FDP in mg which catalyzed the oxidation of 1 μmol of NADH using ε340=6.2 cm−1 mM−1.

Michaelis-Menten parameters for NADH oxidase and NADH:nitric oxide oxidoreductase (NOR) activities were determined at room temperature (~23 °C) in 50 mM MOPS pH 7.3 by measurements of initial rates of NADH consumption (Δ340nm/min) as a function of dioxygen or nitric oxide concentrations. Measurements were carried out in 1-ml septum-capped cuvettes in an N2-filled Vacuum Atmospheres Company glove box using an Ocean Optics USB2000 spectrophotometer. The assay mixtures contained 200 μM NADH, saturating concentrations of NRORtma and Rdtma (4 μM and 10 μM respectively), and FprA2 monomer concentrations of 100 nM (for NADH oxidase) or 40 nM (for NOR). Protein concentrations were determined using ε450 nm = 12,000 M−1cm−1 for both NRORtma and FprA2 monomer and ε492 nm = 8,870 M−1cm−1 for Rdtma [26]. Reactions were initiated by syringe injections of appropriate volumes of either air- or nitric oxide-saturated MOPS buffer. The nitric oxide gas (99.5 %, Praxair) used to prepare the saturated solutions was first passed through a concentrated KOH solution to remove higher oxides of nitrogen [17].

2.5 O2 reduction by FprA1 and FprA2 in the presence of Rd, NROR and NADH

The reduction of O2 was monitored at room temperature (~23°C) as the decrease in the concentration of dissolved O2 vs time under the NADH oxidase assay conditions described above (using C. acetobutylicum proteins) except in a volume of 4 mL using the Fibox3-single-channel fibre-optic oxygen metre (Presens). The operating mode of this device is described elsewhere [27].

3. Results

3.1 Purification and characterization of the C. acetobutylicum FDPs

The C. acetobutylicum genes, cac1027, encoding FprA1, and cac2449, encoding FprA2, were heterologously expressed as N-terminal Strep-tag® fusion proteins in E. coli DH5α and purified in one step by Strep-Tactin® affinity column chromatography [24]. This protocol yielded highly pure FprA1 and FprA2 (Fig. 2) at 2–8 mg l−1 of culture. Gel filtration of the purified recombinant proteins (see Fig. S1) revealed distinct oligomeric structures: a homodimer for FprA1 (99 ± 5 kDa, approximately twice the calculated monomer mass of 48.4 kDa), and homotetramer for FprA2 (191 ± 11 kDa, approximately four times the calculated monomer mass of 49.7 kDa). Storage of FprA2 (48 hours, 4 °C) showed additional peaks at 104 ± 6 kDa and 49 ± 1 kDa corresponding to the formation of dimers and monomers, respectively (data not shown). The purified proteins were bright yellow due to the UV-visible absorption features at 378 nm and 450 nm of the oxidized flavin cofactors, as shown for FprA1 in Fig. 3. TLC analysis identified FMN as the flavin cofactor in both FprA1 and FprA2 (data not shown), as is invariably found in all characterized FDPs together with a diiron site [1518,26,28]. Analyses of FprA2 showed iron/FMN/protein monomer ratios of 2.3/1.0/1.0. Thus, each subunit of these FDPs contains one FMN and one diiron site, i.e., one active site per FprA1/2 monomer.

Fig. 2
SDS-PAGE analysis of heterologous overexpression and purification of C. acetobutylicum Strep-tagged® FprA1, FprA2, NRORcac. Lanes: 1 and 8, molecular weight marker proteins; lanes 2 and 4, crude extracts of induced E. coli DH5α-pI7fprA1 ...
Fig. 3
Visible absorption spectra of oxidized (as-isolated) (——), partially dithionite reduced (- - -) and fully dithionite reduced(— —) C. acetobutylicum FprA1.

3.2 FprA1 and FprA2 function as terminal components of an NADH oxidase

FprA1 and FprA2 (at 1 μM monomer concentrations), by themselves, did not show detectable NADH oxidase activity (data not shown). This result is not surprising, since FDPs from other organisms invariably require oxidoreductase intermediaries for NADH oxidase activity [1518]. The C. acetobutylicum NROR [10] is part of a gene cluster that encodes FprA2. We, therefore, tested whether the purified NROR and Rd could together function as NADH:FprA1 and FprA2 oxidoreductases in NADH oxidase assays. NRORcac alone had very low NADH oxidase activity (<0.2 U mg−1), but this activity drastically increased (>30-fold) when Rdcac, and either FprA1 or FprA2 were added (Fig. 4). This activity was strongly dependent on the presence of all three proteins. In an assay system composed of 0.1 μM NRORcac, 2 μM Rdcac, and 1 μM FprA1 or FprA2 (monomer concentration) NADH was consumed at a maximum rate of 127 ± 9 μmol min−1 per μmol of FprA1 (6.6 U mg−1) and 211 ± 14 μmol min−1 per μmol of FprA2 (11.0 U mg−1). NADPH consumption was not observed when it was substituted for NADH in this assay system. The C. acetobutylicum NROR has previously been shown to efficiently catalyze reduction of Rd by NADH but not by NADPH [6,7]. Thus NROR and Rd function as intermediate components in the electron transfer chain: NADH → NROR → Rd → FprA1/2, in which Rd and not the NROR serves as the proximal electron donor to the FDPs.

Fig. 4
NADH oxidase activity of NRORcac, Rdcac and FprA1 or FprA2. NADH or NADPH consumption was measured as decrease in absorbance at 340 nm. The assay contained 100 μM of NADH, 2 μM of Rdcac, 0.1 μM of NRORcac, and 1 μM of either ...

To verify that FprA1 or FprA2 were indeed acting as the terminal component of NADH oxidase, we directly measured O2 consumption at ~23 °C using an oxygen electrode in solutions corresponding to the assay mixtures of Fig. 4. Thus, NRORcac, Rdcac, and either FprA1 or FprA2 were sequentially added to O2-saturated buffer (~260 μmol l−1). Upon addition of FprA1/2 O2 was consumed at a maximum rate of 51 ± 6 μmol O2 min-1 per μmol of FprA1 and 92 ± 9 μmol O2 min-1 per μmol of FprA2 (Fig. 5). These FDPs could conceivably catalyze reduction of O2 to either H2O or H2O2 according to reactions 1 or 2, respectively.

Fig. 5
O2 consumption by NRORcac, Rdcac and FprA1 or FprA2 in the presence of NADH. The decrease of the O2 concentration was monitored over time using a fibre-optic O2 metre, as described in Material and Methods. The assay mixtures contained initially 250 μM ...
(reaction 1)
(reaction 2)

The mole ratios of NADH oxidation/O2 consumption (127 ± 9 μmol NADH/51 ± 6 μmol O2 = 2.5 for FprA1, and 211 ± 14 μmol NADH/92 ± 9 μmol O2 = 2.3 for FprA2). are much closer to the 2:1 NADH:O2 stoichiometry of reaction 1 than the 1:1 stoichiometry of reaction 2. These results, thus, show that H2O is the predominant product of O2 reduction by both FprA1 and FprA2, as has been invariably observed for other FDPs [1518].

3.3 NADH oxidase vs NADH:nitric oxide oxidoreductase catalytic efficiencies

Because some FDPs function as terminal components of an anaerobic NADH:nitric oxide oxidoreductase (NOR) [17,26,2830], we compared the catalytic efficiencies of the NADH oxidase and NOR activities of FprA2 under conditions of saturating NROR and Rd at ~23 °C. For these determinations the higher-yield and more robust T. maritima NROR (NRORtma) homolog was used for the multiple repetitive initial rate measurements at various O2 or NO concentrations. The data shown in Fig. S2 yielded kcat, Km, and kcat/Km values of 5 s−1, 16 μM, and 3 × 105 s−1M−1, respectively for the NADH oxidase and 34 s−1, 40 μM, and 1 × 106 s−1M−1 for the NOR activities. These values are similar to those reported for other FDPs [26], and indicate that FprA2’s NADH oxidase and NOR catalytic competencies are similar to each other. An improved fit to the NOR activity data (Fig. S2) was obtained using a kinetic model in which the required two nitric oxides per turnover show cooperative binding to the diiron site, as has been observed for other FDPs [17,26]. Such cooperativity is neither expected nor observed for the NADH oxidase activity, which requires only a single O2 per active site per turnover.

4. Discussion

The induction of various types of oxidative stress responses allows anaerobic microbes to colonize habitats that are subject to periodic influxes of O2. C. acetobutylicum is capable of time-limited survival in fully aerobic or microoxic environments [13], and has been shown to rapidly consume molecular O2 from an aerobic environment upon full activation of its PerR regulon [11]. The results reported here show that the two C. acetobutylicum FDPs, FprA1 and FprA2, which are highly expressed in response to O2 exposure [13,14], are able to serve as the terminal component of a reconstituted NADH oxidase, catalyzing the four-electron reduction of molecular oxygen to water. Both FprA1 and FprA2 contain the characteristic FMNand diiron cofactors, the latter of which is the site of O2 reduction in other FDPs [16]. The electron transfer pathway from NADH to O2 (Fig. 6) can be reconstituted using the native C. acetobutylicum oxidoreductase components. The NADH oxidase specific activities of FprA1/2 determined in this work are at least 100-fold higher than that of the revRbr using the same electron transfer components and similar assay conditions [7]. Therefore, although revRbr is also highly induced upon O2 exposure and highly overexpressed in the aerotolerant perR deletion strain [11], its predominant function is most likely as a scavenger of its preferred substrate, H2O2.

Fig. 6
Model for O2 scavenging in C. acetobutylicum. NROR, NADH:rubredoxin oxidoreductase; Rd, rubredoxin; FprA1, flavo-diiron protein 1; FprA2, flavo-diiron protein 2; revRbr, reverse rubrerythrin; red, reduced; ox, oxidised.

Many (although not all) FDPs have been shown to serve as terminal components of anaerobic NADH:nitric oxide oxidoreductases (NORs), thereby scavenging toxic nitric oxide [17,26,2830]. The E. coli FDP homologue, flavorubredoxin, is in fact induced in response to anaerobic nitric oxide exposure [30]. We show in this work that FprA2 can serve as the terminal component of an NOR with a catalytic efficiency comparable to that of its NADH oxidase activity. Future investigations of the two C. acetobutylicum FDPs will include their in vivo roles in scavenging nitric oxide.

Supplementary Material


Appendix A. Supplementary data:

The following supplementary data is available for this article online:

Table S1. Homology of FprA1 and FprA2 form C. acetobutylicum to previously characterized FDPs.


Fig. S1. Analytical gel filtration of Strep-Tactin-purified C. acetobutylicum FprA1 and FprA2.


Fig. S2. Michaelis-Menten plots for NADH oxidase and NOR activities of C. acetobutylicum FprA2.


This work was supported in part by the SysMO project COSMIC (http://www.sysmo.net) to H.B. and R.-J.F. and by NIH grant GM040388 to D.M.K. O.R. was supported by a fellowship of the Graduiertenförderung of the Federal State of Mecklenburg-Vorpommern. A.M. acknowledges financial support from Grant PN-II-Ideas-107/2007 from the Romanian Ministry for Education and Research.


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