Curr Microbiol DOI 10.

1007/s00284-012-0244-y

HdrC2 from Acidithiobacillus ferrooxidans Owns Two IronSulfur Binding Motifs But Binds Only One Variable Cluster Between [4Fe4S] and [3Fe4S]
Yuandong Liu Shuhui Guo Runlan Yu Jiaju Ji Guanzhou Qiu

Received: 20 August 2012 / Accepted: 20 September 2012 Springer Science+Business Media New York 2012

Abstract The heterodisulde reductase complex HdrABC from Acidithiobacillus ferrooxidans was suggested to own novel features that act in reverse to convert the sulfane sulfur of GSnH species (n [ 1) into sulte in sulfur oxidation. The HdrC subunit is potentially encoded by two different highly upregulated genes sharing only 29 % identity in A. ferrooxidans grown in sulfur-containing medium, which were named as HdrC1 and HdrC2, respectively and had been conrmed to contain ironsulfur cluster by expression and characterization, especially the HdrC1 which had been showed to bind only one [4Fe4S] cluster by mutations. However, the mutations of the HdrC2 remain to be done and the detailed binding information of it is still unclear. Here, we report the expression, mutations, and molecular modeling of the HdrC2 from A. ferrooxidans. This HdrC2 had two identical motifs (Cx2Cx2Cx3C) containing total of eight cysteine residues potentially for ironsulfur cluster binding. This puried HdrC2 was exhibited to contain one variable cluster converted between [4Fe4S] and [3Fe4S] according to different conditions by the UV-scanning and EPR spectra. The site-directed mutagenesis results of these eight residues further conrmed that the HdrC2 in reduction with Fe2? condition loaded only one [4Fe4S]? with spin S = 1/2 ligated by the residues of Cys73, Cys109, Cys112, and Cys115; the HdrC2 in natural aeration condition lost the Fe atom ligated by the residue of Cys73 and loaded only one [3Fe4S]0 with spin S = 0; the HdrC2 in oxidation

condition loaded only one [3Fe4S]? with spin S = 1/2. Molecular modeling results were also in line with the experiment results.

Introduction Acidithiobacillus ferrooxidans is a Gram-negative and chemolithoautotrophic c-proteobacterium that obtains energy from the oxidation of various reduced inorganic sulfur compounds (RISCs) and ferrous iron at very acidic pH [1, 2]. It can be used for recovering metals such as copper, gold, uranium, and zinc from low-grade mineral resources and mining wastes [3]; remedying miningimpacted environments [4] and purifying gases containing sulfur compounds [5], so it is very important for industry and has been studied as the type bacterium in leaching environment. The heterodisulde reductase complex HdrABC in sulfate reducing archaea and bacteria [6] and methanogenic archaea [7] catalyzed the reversible reduction of the disulde bond X-S-S-X coupled with energy conservation, and was composed of three subunits, the avoprotein HdrA, the disulde action HdrB, and the ferredoxin-like HdrC. A gene cluster HdrC2-hdrB1-hdrA-orf2-hdrC2hdrB2 (gene locus AFE_25502555) and a gene hdrB (AFE_2586) in A. ferrooxidans ATCC 23270 potentially encoding the HdrABC complex were found and they were also highly upregulated in cells grown in sulfur-containing medium [8, 9]. However, the HdrABC complex in A. ferrooxidans was predicted to have novel features that work in reverse to catalyse the substrate, sulfane sulfur of GSnH species (n [ 1) and most likely GSSH, into sulte in the RISCs oxidation [8, 10]. So, detailed experimental efforts must be done to further investigate this HdrABC and the

Y. Liu (&) S. Guo R. Yu J. Ji G. Qiu Key Lab of Biometallurgy of the Ministry of Education of China, School of Minerals Processing and Bioengineering, Central South University, Biobuilding, Lushan South Road 932, Yuelu District, Changsha 410083, Hunan Province, Peoples Republic of China e-mail: yuandong_liu@sina.cn

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involved genes. There are totally two different genes hdrC1 (AFE_2555) and hdrC2 (AFE_2551) sharing only 29 % identity potentially encoding the HdrC subunit in A. ferrooxidans, which were named as HdrC1 and HdrC2, respectively and had been conrmed to contain ironsulfur cluster by expression and characterization, especially the HdrC1 which had been showed to bind only one [4Fe4S] cluster by mutations [11, 12]. However, the mutations of the HdrC2 remain to be done and the detailed binding information of it is still unclear. Here, we report the expression, mutations, and molecular modeling of the HdrC2 from A. ferrooxidans ATCC 23270. The obtained recombinant HdrC2 was demonstrated to bind only one variable cluster reversibly converted from [4Fe4S]1?/2? to [3Fe4S]0/1? according to the different conditions of aeration, oxidation, and reduction with Fe2?, respectively.

made in Germany. The amplication products were analyzed by electrophoresis on a 0.9 % agarose gel and stained with ethidium bromide. The resulting PCR product was gel puried, double digested, and ligated into a pET-28a expression vector resulting in the pET-28a::HdrC2 plasmid [13]. This vector had been designed for heterologous gene expression in E. coli, and produces a protein with a tag of six histidines at the amino terminus, which aided on its purication. The constructed pET-28a::HdrC2 plasmid was transformed into DH5a competent cells for screening purposes. The isolated pET-28a::HdrC2 plasmid was then transformed into E. coli strain BL21(DE3) competent cells for expression purposes. Construction of A. ferrooxidans HdrC2 Mutant Plasmids A QuikChange mutagenesis kit (Stratagene) was applied for constructing eight of the pHdrC2(C63A, C66A, C69A, C73A, C109A, C112A, C115A, and C119A) mutant expression plasmids. The plasmid pET-28a::HdrC2 was used as a template for constructing mutant sequence through gene splicing by overlap extension (SOE) PCR reaction. The following primer and their antisense primer were synthesized to introduce the mutated sequence: C63A, C63A-F(forward): 50 -CGAGAACGTGCACCGTG CCTGGCAGTGCGGCT-30 , codon TGC for cysteine (C) was changed to codon GCC for alanine (A); C63AR(reverse): 50 -AGCCGCACTGCCAGGCACGGTGCACG TTCTCG-30 ; C66A, C66A-F(forward): 50 -GTTGCTGGCA GGCCGGCTCCTGCACGAATT-30 ; codon TGC for cysteine (C) was changed to codon GCC for alanine (A), C66A-R(reverse): 50 -AATTCGTGCAGGAGCCGGCCTG CCAGCAAC-30 ; C69A, C69A-F(forward): 50 -TCCTGC ACGAATTCCGCCACCATGTATGCGC-30 ; codon TGC for cysteine (C) was changed to codon GCC for alanine (A), C69A-R(reverse): 50 -GCGCATACATGGTGGCGGA ATTCGTGCAGGA-30 ; C73A, C73A-F(forward): 50 -CTC CTGCACGAATTCCGCCACCATGTATGCG-30 , codon TGC for cysteine (C) was changed to codon GCC for alanine (A), C73A-R(reverse): 50 -CGCATACATGGTGGCGGAATTCGTG CAGGAG-30 ; C109A, C109A-F(forward): 50 -CATTATCTGGCAGGCTGTTTCGTGTAACAAG-30 , codon TGT for cysteine (C) was changed to codon GCT for alanine (A), C109A-R (reverse): 50 -CTTGTTACACGAAACAGCCTGCCAGATAAT G-30 ; C112A, C112A-F(forward): 50 -AGTGTGTTTCGGCTAACAAGTGCACCAAT-30 , codon TGT for cysteine (C) was changed to codon GCT for alanine (A), C112A-R(reverse): 50 -AT TGGTGCACTTGTTAGCCGAAACACACT-30 ; C115A, C115 A-F(forward): 50 -CGTGTAACAAGGCCACCAATATCTGCC CCAAG-30 , codon TGC for cysteine (C) was changed to codon GCC for alanine (A), C115A-R: 50 -CTTGGGGCAGATATTGG TGGCCTTGTTACACG-30 ; C119A, C119A-F(forward): 50 -AC

Materials and Methods Materials A. ferrooxidans ATCC23270 was obtained from the American type culture collection. A HiTrap chelating metal-afnity column was purchased from GE healthcare LTD. DH5a competent cells, E. coli strain BL21(DE3) competent cells came from Invitrogen Life Technologies. The Plasmid Mini kit, a gel extraction kit and synthesized oligonucleotides were obtained from Sangon Company of Shanghai. Taq DNA polymerase, T4 DNA ligase, and restriction enzymes came from MBI Fermentas of Germany. All other reagents were of research grade or better and were obtained from commercial sources. Cloning of the hdrC2 Gene from A. ferrooxidans Genomic DNA from A. ferrooxidans ATCC 23270 was prepared using the EZ-10 spin column genomic DNA isolation kit from Bio Basic Inc., according to the manufacturers instructions for bacterial DNA extraction. This genomic DNA was used as a template for polymerase chain reaction (PCR) reaction. The gene was amplied by PCR using the following primers. The forward primer (HdrC2-F) was 50 -CGCGCGGATCCGCTATTCATGAAAAAACAC TGATC-30 , containing a BamHI site (GGATCC). The reverse primer (HdrC2-R) was 50 -CTGCAGAAGCTTTT AGCCATGATGCGCGG-30 , containing a HindIII site (AAGCTT), a stop anticodon. PCR amplication was performed using Taq DNA polymerase, and samples were subjected to 30 cycles of 45 s of denaturation at 95 C, 45 s of annealing at 55 C, and 2 min of elongation at 72 C in a Mastercycler Personal of Eppendorf Model

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AAGTGCACCAATATCGCCCCCAAGGACGT-30 , codon TGC for cysteine (C) was changed to codon GCC for alanine (A), C119A-R(reverse): 50 -ACGTCCTTGGGGGCGATATTGGTGCACTTGT-30 . The obtaining of mutant sequence required three different PCR process. The steps of obtaining C63A mutant sequence: First, used HdrC2-F&C63A-R as the primer, pET-28a::HdrC2 as the template, PCR amplication was performed using PfuDNApolymerase samples were subjected to 30 cycles of 45 s of denaturation at 95 C, 45 s of annealing at 55 C, and 90 s of elongation at 72 C in a Mastercycler Personal of Eppendorf Model made in Germany, products sighed E1; Second, PCR used C63AF&HdrC2-R as primer, pET-28a::HdrC2 as the template, PCR amplication was performed using PfuDNApolymerase samples were subjected to 30 cycles of 45 s of denaturation at 95 C, 45 s of annealing at 55 C, and 90 s of elongation at 72 C in a Mastercycler Personal of Eppendorf Model made in Germany. Products signed E2; Third, PCR as HdrC2-F&HdrC2-R as primer, E1&E2 as the template, PCR amplication was performed using PfuDNApolymerase samples were subjected to 30 cycles of 45 s of denaturation at 95 C, 45 s of annealing at 55 C, and 2 min of elongation at 72 C in a Mastercycler Personal of Eppendorf Model made in Germany. The resulting PCR product was gel puried, double digested and ligated into a pET-28a expression vector resulting in the pHdrC2(C63A) mutant plasmid. Also, the other seven mutant plasmids were obtained by the same method of pHdrC2(C63A). The constructed mutant plasmids were transformed into DH5a competent cells for screening purposes. The isolated mutant plasmids were then transformed into E. coli strain BL21(DE3) competent cells for expression. Expression of Recombinant HdrC2 Wild-Type and its Mutant Proteins in E.coli The E. coli strain BL21(DE3) cells with pET-28a::HdrC2 plasmid or mutant plasmids were grown at 25 C in 800 ml of TB medium containing kanamycin (100 mg/l) to an OD600 of 0.6. At this point, the cells were shaken at 180 rpm at room temperature with 0.6 mM IPTG overnight. The culture was centrifuged and the cells were washed with an equal volume of sterile water. The cells were harvested by centrifugation at 45009g, 4 C for 10 min. The protein extraction from the inclusion bodies was performed in the following steps: (1) the cell pellet was suspended in lysis buffer 1 (50 mM TrisHCl, 1 mM EDTA, 0.5 M NaCl and 5 mg lysozyme, pH 8.0) at room temperature for half an hour, then sonicated for 15 min and centrifuged; the cell pellet obtained in (1) was suspended in

washing buffer (50 mM TrisHCl, 0.5 M NaCl, 2 % triton100 and 2 M Urea, pH 8.0) at room temperature for 15 min, centrifuged at 120009g, 4 C for 15 min, repeated that process one time more. (3) extraction of the recombinant protein was carried out in the presence of 8 M urea, magnetic stirring, 4 C for 3 h (4) dialyze the substance of (3) buffer 1 (20 mM TrisHCl, 150 mM NaCl, 2 mM Reduced Glutathione, 0.02 mM Oxidized Glutathione, 5 % glycerol, 0.005 % Tween, 3 M Urea) & buffer 2 (Same as Buffer 1 except 1 M Urea) & buffer 3 (20 mM TrisHCl, 150 mM NaCl, 5 % glycerol) in sequence, 4 C for 8 h. Purication of the HdrC2 from A. ferrooxidans The Hi-Trap column was rst equilibrated with 0.1 M nickel sulfate to charge the column with nickel ions followed by ve column volumes of MiliQ water to remove unbound nickel ions from the column, and then by ve column volumes of start buffer (20 mM potassium phosphate, pH 8.0, 0.5 M NaCl) to equilibrate the column. The claried sample was applied to the Hi-Trap column after ltering it through a 0.45 lm lter. The column was washed with ve column volumes of start buffer followed with ve column volumes of wash buffer (20 mM potassium phosphate, pH 8.0, 0.5 M NaCl, 50 mM imidazole), and subsequently the protein was eluted with an elution buffer (20 mM potassium phosphate, pH 8.0, 0.5 M NaCl, 500 mM imidazole). The method of Bradford was used to determine the protein content with BSA as the standard. The eluted fractions were analyzed by SDSpolyacrylamide gel electrophoresis (SDS-PAGE) with 12 % of acrylamide. The gels were stained with Coomassie Brilliant Blue R-250. The puried enzyme fractions were combined and dialyzed against a 20 mM potassium phosphate buffer, pH 6.0, 5 % glycerol, and 5 mM b-mercaptoethanol, and then stored in a -80 C freezer. The aerated purication conditions used in this work may make the FeS cluster to reversibly change by oxidation and loss of some ions of iron and sulfur, so the protein was further investigated by being incubated anaerobically at 25 C in a reducing medium with 5-fold molar excess of both Na2S and Fe(NH4)2(SO4)2 for 3 h in the presence of 5 mM dithiothreitol in 0.1 M TrisHCl, pH 8.0, and then desalted on Sephadex G-25 column to reconstitute the FeS cluster. The oxidized HdrC2 was obtained by incubating with 5 mM ammonium persulfate for 30 min. UVVis Scanning and EPR Spectra UVVisible spectrum scanning was carried out at 25 C on a Techcomp UV-2300 spectrophotometer. The samples of naturally aerated, reduced and oxidized HdrC2 proteins

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(10 lM) were prepared in 20 mM phosphate buffer containing 0.5 M NaCl, pH 6.0. X-band electronic paramagnetic resonance (EPR) spectra were recorded at 100 K on a JEOL JES-FE1XG spectrometer. Parameters for recording the EPR spectra were typically 1530 mT/min sweep rate, 0.63 mT modulation amplitude, 9.14 GHz frequency, and 4 mW incident microwave power, sweep time was 2 min. The samples were diluted to 5 lM in 20 mM phosphate buffer containing 0.5 M NaCl, pH 8.0.

Molecular Modeling of the HdrC2 from A. ferrooxidans All molecular modeling were performed with Insight II software package developed by Accelrys software Inc, running on the Dell Precision 470 workstation with Red Hat Linux system. The rst step was to search for the protein data bank (PDB) of RCSB to nd related protein structures as templates. The second step was to build the initial 3D structures of this protein. The program Modeler was performed to model the structure of this protein based on these templates, which had none prosthetic group and corresponded to the apoprotein of the HdrC2. Then, the possible binding prosthetic groups were added to it to form all kind of the possible initial holoprotein models of the HdrC2 by manual. For the third step, all of these initial models were respectively encompassed by a 2 nm layer of solvent water molecules to be optimized through a series of molecular simulation procedures. For the nal step, the obtained optimized models were assessed by ProStat and Prole3D [14].
Fig. 1 Characterizations of HdrC2 proteins from A. ferrooxidans. a From left to right, the denatured HdrC2 protein wild-type, the recovered HdrC2 protein wild-type, the recovered HdrC2 mutant C63A, HdrC2 mutant C109A, H2O. b UVVisible spectra, from bottom to top, the dashed and dot lane indicates the C109A mutant; the dot lane indicates the wild-type in aeration; the dashed lane indicates the wild-type in oxidation; the solid lane indicates the wildtype in reduction with Fe2?. c EPR spectra, from bottom to top, the C109A mutant, the wild-type in aeration, the wild-type in oxidation, the wild-type in reduction with Fe2?. (The results of the recovered HdrC2 mutant C63A, C66A, C69A, and C119A were identical to that of wild-type; the results of the recovered HdrC2 mutant C112A and C115A were identical to that of C109; the results of the recovered HdrC2 mutant C73A in aeration or oxidation was identical to that of wild-type, the results of the recovered HdrC2 mutant C73A in reduction with Fe2? was identical to itself in aeration; so all these data not shown)

Results and Discussion Expression and Purication of HdrC2 Wild-Type and it Mutant Proteins PCR technique was used to successfully add restriction enzyme cutting site to the terminal of the HdrC2 from A. ferrooxidans. Mutant expression plasmids of pHdr C2(C63A, C66A, C69A, C73A, C109A, C112A, C115A, and C119A) were constructed, and their sequences were veried for the presence of the directed mutation and the absence of SOE PCR-generated random mutations by DNA sequencing. The best protein expression levels were obtained under induction with 0.8 mM IPTG at 25 C, overnight. However, the recombinant proteins were expressed in inclusion bodies in all conditions tested, at the different temperatures. The inclusion bodies were washed, separated from the cells, and solubilized by urea solution or ammonium carbonate, and then renatured by dilution and dialysis. The fraction and solution containing the wild-type

protein displayed a dark brown color (Fig. 1a), which indicates the presence of FeS clusters. Nickel metalafnity resin columns were used for single-step purications of His-tagged A. ferrooxidans HdrC2 wildtype and mutant proteins. The eluted wild-type HdrC2 in natural aeration was observed to be a light brown protein (Fig. 1a),

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indicating the FeS cluster was still bound to the protein after purication. These results were in perfect agreement with previously reported the HdrC2 from A. ferrooxidans strain LR [11]. When incubated respectively in oxidation or in reduction and Fe2?, the puried wild-type HdrC2 showed no change in color with naked-eye observation. The HdrC2 mutant proteins were also puried following the same way as wild-type protein and obtained as soluble proteins. The purity of HdrC2 wild-type and the mutant proteins were examined by SDS-PAGE and single bands corresponding to the 28.2 kDa proteins were observed with [95 % purity, which was in agreement with the deduced molecular mass of a monomer.

Eight Conserved Cysteines Bind Only One Variable Cluster Between [4Fe4S] and [3Fe4S] It was showed that the HdrC2 from A. ferrooxidans harbored two copies of motifs (Cx2Cx2Cx3C) potentially for FeS cluster binding, the rst motif contained Cys63, Cys66, Cys69, and Cys73, the second motif contained Cys109, Cys112, Cys115, and Cys119. Sequence alignment for HdrC from various sources also showed that these eight cysteines in the HdrC2 were highly conserved residues [12]. To further investigate the HdrC2 how to bind FeS cluster, eight mutations respectively for these cysteine residues of HdrC2 from A. ferrooxidans were rst created, and their mutant plasmids were expressed in E. coli, puried by afnity chromatography to homogeneity, and then detected by UVVis scanning and EPR spectrum under the different conditions of natural aeration, oxidation, and reduction, respectively. As shown in Fig. 1 and Table 1, the results of Cys63, Cys66, Cys69, and Cys119 mutant proteins were completely identical to that of the wild-type protein in all tested conditions; the result of Cys73 mutant protein was also completely identical to that of the wild-type protein under the conditions of aeration and oxidation, but showed difference to that of the wild-type protein under the condition of reduction, which owned the same appearance just like itself under the condition of aeration; however, those of Cys109, Cys112, and Cys115 mutant proteins showed no color, no absorptions between 300 and 500 nm comparing to that of wild-type HdrC2, and no ERP signal, indicating the absence of the FeS cluster in these mutant proteins. These results strongly suggested that the eight conserved cysteines in the HdrC2 from A. ferrooxidans bound only one FeS cluster, respective removal of the sulfhydryl group of Cys109, Cys112, and Cys115 resulted in the FeS cluster loss, but respective removal of the sulfhydryl group of Cys63, Cys66, Cys69, and Cys119 had no inuence on the FeS cluster, removal of the sulfhydryl group of Cys73 caused the protein bind only [3Fe4S] cluster in all conditions. Based on these mutation results, we can rmly conrm that the HdrC2 from A. ferrooxidans owns eight conserved cysteines but binds only one variable cluster, which exhibited the [4Fe4S] form ligated by the residues of Cys73, Cys109, Cys112, and Cys115 in reduction and Fe2? condition, but the [3Fe4S] form with the Fe atom ligated by the residue of Cys73 loss in aeration and oxidation conditions. So, the HdrC2 from A. ferrooxidans binds the variable cluster centre with the [4Fe4S]2?/1? reversibly converted into a [3Fe4S]1?/0 according to different conditions. The variation relationships of structures, oxidation states, spin states, and conditions of the clusters of [3Fe4S] and [4Fe4S] in the HdrC2 were also proposed as shown in

UV-scanning and EPR Spectra of the HdrC2 from A. ferrooxidans The UVVisible spectra of aerated, oxidized and reduced forms for the recombinant HdrC2 were shown in Fig.1b. The major visible absorption peaks of the aerated and oxidized forms were all located around at 330 and 410 nm respectively, but the oxidized form exhibited an increase in the absorbance compared to the aerated form, which were also well in line with the previously reported spectra of the ironsulfur cluster-containing protein of HdrC2 from A. ferrooxidans strain LR [11]. The major visible absorption peaks of the reduced form were located around at 330 and 420 nm, which were, however, similar to the previously reported spectra of the ironsulfur cluster-containing protein of HdrC1 from A. ferrooxidans strain ATCC 23270, the [4Fe4S] ferredoxin from Desulfovibrio desulfuricans, respectively [12, 15]. In addition, the whole spectrum of the reduced form showed a higher absorbance and a slight shift for one of the major peaks compared to those of the aerated and oxidized forms. So it was conrmed that the wild-type HdrC2 in the three investigated conditions all loaded the FeS cluster. The EPR spectra of aerated, oxidized, and reduced forms for the recombinant HdrC2 were shown in Fig. 1c. The HdrC2 in natural aeration showed an EPR silence. The HdrC2 in oxidation exhibited only an EPR signal with g value of 2.02, which was also well in line with the result of the HdrC2 from A. ferrooxidans strain LR [11] and showed the typical characteristic of the proteins harboring the [3Fe4S]?1 with spin S = 1/2. However, the EPR spectrum of the HdrC2 in reduction and Fe2? exhibited a rhombic signal with g values of 2.06, 1.94, and 1.89, which are similar to those of the partially reduced 8Fe ferredoxins which contain a singly reduced and non-interacting [4Fe4S]?1 with spin S = 1/2 [16]. However, the HdrC2 from A. ferrooxidans how to bind FeS cluster remained to be conrmed.

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L. Yuandong et al.: HdrC2 from Acidithiobacillus ferrooxidans Binds Only One Variable Cluster Table 1 Characterizations of the puried HdrC2 and its mutant proteins under different conditions Puried HdrC2 proteins Incubated with ammonium persulfate Color Wild-type C63A C66A C69A C73A C109A C112A C115A C119A Brown Brown Brown Brown Brown White White White Brown UVVis peaks (nm) 330, 410 330, 410 330, 410 330, 410 330, 410 No No No 330, 410 EPR (g) 2.02 2.02 2.02 2.02 2.02 0 0 0 2.02 Aeration Color Brown Brown Brown Brown Brown White White White Brown UVVis peaks (nm) 330 330 330 330 330 No No No 330 EPR (g) 0 0 0 0 0 0 0 0 0 Incubated anaerobically with both Fe(NH4)2(SO4)2 and Na2S Color Brown Brown Brown Brown Brown White White White Brown UVVis peaks (nm) 330, 420 330, 420 330, 420 330, 420 330 No No No 330, 420 EPR (g) 2.06, 1.94, 1.89 2.06, 1.94, 1.89 2.06, 1.94, 1.89 2.06, 1.94, 1.89 0 0 0 0 2.06, 1.94, 1.89

Fig. 2. Just like the HdrC1 from A. ferrooxidans, the reason for the HdrC2 with one FeS cluster loss could be explained by the novel features of the HdrABC in A. ferrooxidans working in reverse to catalyse the oxidation of disulde intermediaries to sulte [8]. One FeS cluster loss in the HdrC2 may rightly contribute to cause the electrons reverse transfer, as described for the cytochrome bc1 complex and the NADH1 dehydrogenase complex of the uphill electrons transfer components in A. ferrooxidans [2, 8, 17]. Molecular Structure Modeling of the HdrC2 from A. ferrooxidans When searched in the PDB with the sequence of the HdrC2, no obvious similar sequence of the deposited structures was found. However, the structure of the iron sulfur protein subunit of fumarate reductase from E. coli (PDB code 1L0V) [18] had the highest identity 24 % with the sequence of the HdrC2 from A. ferrooxidans in the PDB search result, so the PDB 1L0V was selected as the template to build the HdrC2 molecular model and an initial apoprotein molecular model without FeS cluster binding of the HdrC2 was created through homology modeling procedure. In this initial apoprotein model, the eight cysteine residues were obviously divided into two spacial groups. However, the distribution of the eight cysteine residues in the two groups did not correspond to that in the two motifs, the rst group included Cys63, Cys66, and Cys69 in the rst motif and Cys119 in the second motif, the second group included Cys109, Cys112, and Cys115 in the second motif and Cys73 in the rst motif (Fig. 3). This situation also exists in the 2[4Fe4S] ferredoxin [19], the

FeS subunits of bacterial fumarate reductase [18] and so on. To further detect the HdrC2 how to bind FeS cluster, two possible holoprotein molecular models of HdrC2 respectively corresponding to the [4Fe4S] ligated by the residues of Cys73, Cys109, Cys112, and Cys115 and the [3Fe4S] with the Fe-atom ligated by the residue of Cys73 loss were constructed based on the initial apoprotein model. These models were then respectively encompassed by a layer of solvent water molecules to be optimized through a series of molecular simulation procedures. The obtained optimized holoprotein models were nally assessed by Prole3D and ProStat programs and indicated to be reliable (Fig. 3). In the structure model of the HdrC2 harboring [4Fe4S] (Fig. 3b), the length of the bond ligated

Fig. 2 Variation relationships of structures, oxidation states, spin states, and conditions of the FeS cluster in the HdrC2 from A. ferrooxidans converted between [3Fe4S] and [4Fe4S]

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Fe-atom ligated by the residue of Cys73 and loaded only one [3Fe4S]0; the HdrC2 in oxidation condition loaded only one [3Fe4S]?. Molecular modeling results were also well in line with the experiment results.
Acknowledgments This study was supported by the National Natural Science Foundation of P. R. China (50904080 and 51274268) and the National Basic Research Program of P. R. China (2010CB630900).

References
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Fig. 3 Molecular structure modeling of the HdrC2 from A. ferrooxidans. a The structure model of the HdrC2 in natural aeration or oxidation conditions which loaded a [3Fe4S] cluster ligated by Cys109, Cys112, and Cys115. b The structure model of the HdrC2 in reduction with Fe2? condition which loaded a [4Fe4S] cluster ligated by Cys73, Cys109, Cys112, and Cys115 with the bond length of 2.62, 2.28, 2.33, and 2.23 A, respectively

between [4Fe4S] and Cys73 was obviously longer than that of other three residues of Cys109, Cys112, and Cys115, respectively, which indicated that the ligated strength of Cys73 was looser than that of Cys109, Cys112, and Cys115, respectively and explained that the Fe-atom ligated by Cys73 in [4Fe4S] was easy to rst lose which cause the change of the FeS cluster from [4Fe4S] to [3Fe4S] in the HdrC2. These structural modeling results were in well agreement with those of the experiment results. In summary, we here report the expression, mutations, and molecular modeling of the HdrC2 from A. ferrooxidans. This puried HdrC2 exhibited to contain one variable cluster reversibly converted between [4Fe4S] and [3Fe4S] according to different conditions by the UV-scanning and EPR spectra. The site-directed mutagenesis results of eight key cysteine residues further conrmed that the HdrC2 in reduction with Fe2? condition loaded only one [4Fe4S]? ligated by the residues of Cys73, Cys109, Cys112, and Cys115; the HdrC2 in natural aeration condition lost the

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L. Yuandong et al.: HdrC2 from Acidithiobacillus ferrooxidans Binds Only One Variable Cluster 15. Pedro MR, Isabel M, Anjos LM, Jose JGM (2003) Spectroscopic characterization of a novel 2[4Fe4S] ferredoxin isolated from Desulfovibrio desulfuricans ATCC 27774. Inorg Chim Acta 356:7 16. Prince RC, Adams MW (1987) Oxidation-reduction properties of the two Fe4S4 clusters in Clostridium pasteurianum ferredoxin. J Biol Chem 262(11):51255128 17. Brasseur G, Bruscella P, Bonnefoy V, Lemesle-Meunier D (2002) The bc(1) complex of the iron-grown acidophilic chemolithotrophic bacterium Acidithiobacillus ferrooxidans functions in the reverse but not in the forward direction. Is there a second bc(1) complex? Biochim Biophys Acta 1555(13):3743 18. Iverson TM, Luna-Chavez C, Croal LR, Cecchini G, Rees DC (2002) Crystallographic studies of the Escherichia coli quinolfumarate reductase with inhibitors bound to the quinol-binding site. J Biol Chem 277:1612416130 19. Fukuyama K, Matsubara H, Tsukihara T, Katsube Y (1989) Structure of [4Fe4S] ferredoxin from Bacillus thermoproteo lyticus rened at 2.3 A resolution. Structural comparisons of bacterial ferredoxins. J Mol Biol 210(2):383398

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