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Applied and Environmental Microbiology, April 2009, p. 2439-2445, Vol. 75, No. 8
0099-2240/09/$08.00+0     doi:10.1128/AEM.01325-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Periplasmic c Cytochromes and Chlorate Reduction in Ideonella dechloratans

Anna Smedja Bäcklund,¹ Jan Bohlin,¹ Niklas Gustavsson,^2, and Thomas Nilsson^1*

Department of Chemistry and Biomedical Sciences, Faculty of Technology and Science, Karlstad University, SE-651 88 Karlstad, Sweden,¹ Department of Biochemistry, Lund University, SE-221 00 Lund, Sweden²

Received 13 June 2008/ Accepted 11 February 2009

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
The aim of this study was to clarify the pathway of electron transfer between the inner membrane components and the periplasmic chlorate reductase. Several soluble c-type cytochromes were found in the periplasm. The optical difference spectrum of dithionite-reduced periplasmic extract shows that at least one of these components is capable of acting as an electron donor to the enzyme chlorate reductase. The cytochromes were partially separated, and the fractions were analyzed by UV/visible spectroscopy to determine the ability of donating electrons to chlorate reductase. Our results show that one of the c cytochromes (6 kDa) is able to donate electrons, both to chlorate reductase and to the membrane-bound cytochrome c oxidase, whereas the roles of the remaining c cytochromes still remain to be elucidated. Peptide extracts of the c cytochromes were obtained by tryptic in-gel digestion for matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis. Peptide sequences obtained indicate that the 6-kDa cytochrome c protein is similar to c cytochromes from the chlorate-reducing bacterium Dechloromonas aromatica.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Oxyanions of chlorine (ClO₃^– and ClO₄^–) occur in the environment mainly as by-products from human activities (6, 7). The decomposition of chlorate by microbial respiration is important in the treatment of industrial effluents and has been known since the beginning of the 20th century (2). One of the chlorate-respiring bacteria, the gram-negative Ideonella dechloratans, was isolated by Malmqvist and coworkers (8).

Chlorate metabolism takes place in the periplasmic space between the inner and outer membranes and involves the soluble enzymes chlorate reductase and chlorite dismutase. The reaction takes place in two steps. First, chlorate is reduced to chlorite by chlorate reductase in a two-electron transfer reaction. The second step is the decomposition of chlorite into chloride ions and molecular oxygen, which is catalyzed by chlorite dismutase. Both enzymes have been isolated and characterized, and their genes have been sequenced (4, 5, 15). Chlorate reduction is coupled to cell growth, suggesting that chlorate reductase is part of a respiratory chain that generates an electrochemical gradient, which can serve as the driving force for ATP synthesis. The aim of this study was to investigate the pathways of electron transfer, in particular the route between membrane-bound components of the respiratory chain and the soluble periplasmic enzymes, in I. dechloratans. One interesting aspect is the finding that a gene encoding a soluble c-type cytochrome is located downstream of the gene for chlorate reductase (GenBank accession no. EU768872) (J. Bohlin, A. Smedja Bäcklund, N. Gustavsson, S. Wahlberg, and J. Nilsson, unpublished data).

Although the electron transport pathways in bacteria differ, two major strategies for the transfer of electrons to soluble enzymes seem to occur. One strategy is the oxidation of quinol by cytochrome bc₁ complex, followed by electron transfer to a soluble c-type cytochrome. In the other strategy, where the bc₁ complex is absent or not involved, electron transfer is mediated by a membrane-anchored periplasmic c-type cytochrome belonging to the NapC/NirT family (13).

The chlorate reductase in I. dechloratans shows similarity to molybdopterin-containing members of the type II subgroup of the dimethyl sulfoxide reductase family (10). One member of the family, dimethyl sulfoxide dehydrogenase (Ddh) from the phototrophic Rhodovulum sulfidophilum, utilizes a soluble cytochrome c for transfer of electrons, but in the reverse direction. The β subunit in Ddh donates electrons to the membrane-bound photochemical center, mediated by the soluble cytochrome c₂ (9). Another member of the dimethyl sulfoxide reductase family, the closest known relative to chlorate reductase in I. dechloratans, is selenate reductase from Thauera selenatis (14). The quaternary structure of this enzyme is very similar to that of Ddh in R. sulfidophilum, and it has been suggested that the enzyme may interact with a periplasmic c cytochrome that receives electrons from the bc₁ complex (10). Several other (per)chlorate-reducing bacteria, such as Dechloromonas agitata (1), Dechloromonas aromatica strain RCB (3), and strain GR-1 (12), have been isolated. In D. aromatica, several genes encoding NapC/NirT-like cytochromes have been found, but the physiological roles of the corresponding proteins are not known (3). The electron transfer pathways in D. agitata and strain GR-1 are unknown.

The present study aims at investigating the role of soluble c-type cytochromes as electron mediators between the bc₁ complex in the inner membrane and the periplasmic chlorate reductase in I. dechloratans. We have found that at least one of the periplasmic c-type cytochromes is capable to act as a electron donor to the enzyme chlorate reductase.

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Extraction and identification of soluble c cytochromes.
Ideonella dechloratans was obtained from the Culture Collection of Göteborg University, Göteborg, Sweden. Cells were cultured anaerobically, and periplasmic proteins were extracted by osmotic shock as previously described (15). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using NuPAGE 4 to 12% bis-Tris gel (Invitrogen, Lidingö, Sweden) with 2-(N-morpholino)ethanesulfonic acid (MES) running buffer (Invitrogen, Lidingö, Sweden); a reducing agent was not used. Gels were stained using 3-,3'-,5-,5'-tetramethylbenzidine for detection of heme-containing proteins by the method of Thomas et al. (16).

Protein purification.
The periplasmic extract was adjusted with MES to pH 6.5 and loaded onto a HiTrap SP FF, low subcolumn (5 ml) (GE Healthcare, Uppsala, Sweden) at a flow rate of 5 ml/min. The column was washed with 50 mM MES (pH 6.5), and the sample was eluted using a gradient of 0 to 1 M NaCl in a total volume of 30 ml. A fraction containing 6-kDa cytochrome c (eluted before the gradient was started) was concentrated with Amicon Ultra-4 centrifugal filter units (3,000-Da cutoff). Fractions containing the 10-kDa cytochrome (eluted between 15 and 50 ml NaCl) were pooled, filtered with Amicon Ultra-15 centrifugal filter units (50,000-Da cutoff), and concentrated with Amicon Ultra-15 centrifugal filter units (5,000-Da cutoff). The flowthrough fraction was dialyzed against 20 mM Tris-HCl (pH 8.5) at 4°C applied to a Q FF column (5 ml) and eluted using increasing sodium chloride (0 to 1 M, total gradient volume was 150 ml). All fractions were characterized by NuPAGE (Invitrogen, Lidingö, Sweden).

Redox activity assay.
In order to test the sensitivity of the soluble c cytochromes for spontaneous air oxidation, 2 ml of periplasmic extract was flushed with nitrogen, reduced by 10 µl dithionite (25 mM), and then exposed to air. After 10 min, 25 µl of cell homogenate was added.

Chlorate reductase activity was measured by the enzyme assay described previously (5). The cytochrome c fractions from ion-exchange chromatography were diluted with sodium phosphate (20 mM, pH 7.5) up to 2 ml and mixed with a catalytic amount of chlorate reductase in a quartz cuvette, equipped with a rubber septum. The source of chlorate reductases was periplasmic extract or cell homogenate. The mixture was flushed with nitrogen and stirred for 3 min. Portions of dithionite were injected by a syringe until complete reduction was reached. A small amount of sodium chlorate (final concentration of 5 mM; 1 M stock solution, nitrogen flushed) was added to test for oxidation of the c cytochromes by chlorate reductase. Optical absorption spectra were measured at 400 to 700 nm (UV-1601PC; Shimadzu).

Tryptic in-gel digestion.
Proteins, separated by NuPAGE and stained by SimplyBlue SafeStain (Invitrogen, Lidingö, Sweden), were excised from gels and divided into small pieces. The gel pieces were incubated in 100 µl of 50% ethanol (50 mM NH₄HCO₃ [pH 7.8], 30 min), in 1.5-ml Protein Lo Bind microcentrifuge tubes (Eppendorf, Göteborg, Sweden). The liquid was removed, and the step was repeated once. Ethanol (99.5%; spectrographic grade, 75 µl) was added, and the gel pieces were incubated for 10 min. The ethanol was removed, and remaining ethanol was evaporated with a Speedvac (15 min). Trypsin solution (10 ng/µl in 4.5 µl of 50 mM NH₄HCO₃) was added to the dried gel pieces, which were then incubated on ice. After 30 min, 12 µl of 50 mM NH₄HCO₃ was added to the digestion product, which was then incubated at 37°C overnight. Peptides were extracted by the addition of 12 µl of 0.5% trifluoroacetic acid (TFA), followed by incubation for 30 min. Liquid was removed with a Speedvac, and the dried peptides were stored at –20°C.

Liquid chromatography.
Peptide extracts were separated by nanoflow reversed-phase liquid chromatography using a 1100 Series Nanoflow LC system (Agilent Technologies, Waldbronn, Germany) with mobile phase buffers A (1% [vol/vol] acetonitrile and 0.1% TFA) and B (90% acetonitrile and 0.1% TFA). The peptide extract was first applied to a precolumn (Zorbax 300 SB C₁₈ column [5 by 0.3 mm]) and then separated on a separation column (Zorbax 300 SB C₁₈ column [150 by 0.075 mm]) at a flow rate of 300 nl/min. The eluent from the column was collected in 48 fractions for each sample, directly onto stainless steel matrix-assisted laser desorption ionization (MALDI) targets. The following gradient profile was used: 0 to 9 min, 100% buffer A (isocratic); 10 to 17 min, 10% buffer B (isocratic); 17 to 70 min, 10 to 70% buffer B; 71 to 76 min, 100% buffer B (isocratic); 77 to 82 min, 100% buffer A (isocratic). Fraction collection was performed from 28 min to 61 min in 0.7-min intervals.

MALDI-TOF MS.
All mass spectrometry (MS) analyses were performed using a 4700 Proteomics Analyzer (Applied Biosystems, Framingham, MA) mass spectrometer in positive reflector mode. Peptide extracts were resuspended in 15 to 20 µl of 0.1% TFA, and 0.5 µl of each extract was deposited directly on the matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) sample support. The samples were allowed to dry, and then 0.5 µl of matrix solution (5-mg/ml -cyano-4-hydroxycinnamic acid, 50% acetonitrile, 0.1% TFA, 25 mM citric acid) was added to each sample. For MS and tandem MS (MS-MS) analyses, approximately 2,000 and 5,000 single laser shot spectra were summed up, respectively. Fractions of samples, separated by nanoliquid chromatography, were allowed to dry before the addition of 0.5 µl of matrix solution.

Protein identification.
Protein identification was performed searching the Swiss-Prot, trEMBL, and NCBInr protein sequence databases using an in-house Mascot search engine (version 1.9; Matrix Science, London, United Kingdom). The search parameter settings were as follows: peptide mass tolerance, ±50 ppm; fragment ion mass tolerance, ±0.3 Da; one missed cleavage site; fixed modifications, carbamidomethylation (C) for MS-MS data and ±50 ppm; one missed cleavage site; fixed modifications, carbamidomethylation (C) for MS data.

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Extraction and characterization of soluble c cytochromes.
Soluble proteins were obtained from the periplasm of chlorate-grown I. dechloratans by osmotic shock and cell lysis. Heme-containing proteins were detected by gel electrophoresis, followed by heme staining (16). Figure 1 shows the heme-containing proteins in the periplasm. Five low-molecular-mass c cytochromes were identified. Their molecular masses were estimated to be 20, 10, 9, 8, and 6 kDa. The strongest bands were the 10- and 6-kDa proteins. The bands at 8 and 9 kDa are not always observed as separate bands, as the case in Fig. 1, suggesting that the 8-kDa component is a degradation product. There are also several proteins with higher molecular masses, between 25 and 45 kDa. The 25-kDa band probably originates from heme b in incompletely denatured chlorite dismutase (16).

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FIG. 1. c-type cytochromes in periplasmic extract, stained for heme. SeeBlue Plus2 prestained standards (Invitrogen) were used as the molecular mass standards (lane 1). Five low-molecular-mass c cytochromes were estimated to have molecular masses of 20, 10, 9, 8, and 6 kDa, respectively (lane 2).

To investigate the roles of periplasmic c-type cytochromes in anaerobic respiration, the periplasmic extract was reduced with dithionite, and the effects of respiratory electron acceptors were investigated. The optical spectrum in Fig. 2 shows that about 45% of the c cytochromes reoxidized after the addition of chlorate in the absence of oxygen. The reaction was complete within 1 minute, and no further changes were observed after prolonged incubation. These results show that at least one of the soluble proteins is able to serve as an electron donor to chlorate reductase. The peak at 552 nm in the difference spectrum indicates that soluble c cytochromes participate in the electron donation.

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FIG. 2. Oxidation of c cytochromes in periplasmic extract. The periplasmic extract was treated with nitrogen and reduced by portions of dithionite. After complete reduction, chlorate was added. The black broken line represents the unreduced periplasm as isolated. The black solid line represents the periplasm completely reduced, and the gray line represents the periplasm in a chlorate-oxidized state. The insert shows the reduced-minus-initial difference spectrum. A, change in absorbance.

Partial separation of periplasmic c cytochromes.
In order to investigate the electron transfer to chlorate reductase in more detail, we attempted to separate c cytochromes of the periplasmic extract. Application of the extract to a cation-exchange column resulted in retention of the 10-kDa cytochrome, whereas the other c cytochromes were not adsorbed. Some of the 6-kDa cytochrome was partially retained and emerged as a second peak of the flowthrough fraction (Fig. 3, lane 1) The 10-kDa cytochrome could be eluted with a salt gradient (Fig. 3, lane 5). The remaining heme proteins were found in the material not adsorbed on the column (Fig. 3, lane 2). The flowthrough fraction was applied to an anion-exchange chromatography column, but the proteins were not adsorbed (Fig. 3, lane 4). Our attempts to separate the remaining proteins were unsuccessful.

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FIG. 3. Partially separated c cytochromes. Fractions from ion-exchange chromatography were identified by SDS-PAGE and heme staining. Lane 1, 6-kDa cytochrome c from cation-exchange chromatography (second peak of the flowthrough); lane 2, flowthrough from cation-exchange chromatography containing 6-, 8-, 9-, and 20-kDa c cytochromes; lane 3, SeeBlue Plus2 molecular mass standards (188, 98, 62, 49, 38, 28, 17, 14, 6, and 3 kDa); lane 4, flowthrough from anion-exchange chromatography containing the same protein as in lane 2 (concentrated); lane 5, fraction from cation-exchange chromatography containing the 10-kDa cytochrome c.

Redox activities.
The ability of the cytochrome fractions to serve as electron donors was investigated by UV/visible spectroscopy. In this case, the source of chlorate reductase was a cell homogenate, which also includes membrane components, such as cytochrome c oxidase. In Fig. 4A, the separated 6-kDa cytochrome c was treated with nitrogen and reduced with dithionite. After the addition of chlorate, the protein was completely reoxidized within 1 minute. Figure 4B illustrates the corresponding experiment with the fraction containing the 10-kDa cytochrome c. The spectrum obtained from the dithionite-reduced sample shows two peaks at 552 and 558 nm, indicating the presence of at least two chromophore components. The difference spectrum obtained by subtracting the initial spectrum from that obtained after reduction with dithionite is shown in the insert in Fig. 4B. The 558-nm component dominates the spectral changes produced by dithionite reduction. The addition of chlorate in the presence of chlorate reductase produced no further change in the spectrum, suggesting that none of the components in the sample was able to serve as an electron donor for chlorate reductase. In the corresponding experiment with the fraction containing 6-, 8-, 9- and 20-kDa c cytochromes, complete reoxidation was observed within 1 minute (Fig. 4C).

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FIG. 4. Effect of chlorate or oxygen on the different cytochrome c fractions from ion-exchange chromatography after reduction with dithionite in the presence of catalytic amounts of chlorate reductase and cytochrome c oxidase from a cell homogenate. On panels A to F, the x axis shows wavelength (in nanometers) and the y axis shows absorbance. The black broken lines represent the unreduced state of the fractions as isolated. The black solid lines show the dithionite-reduced state of the fractions. The gray lines show the spectrum obtained after the addition of oxidant. The small inserts show the reduced-minus-initial difference spectra. A, change in absorbance. (A to C) Effect of chlorate on the fractions containing the 6-kDa cytochrome (A), 10-kDa cytochrome (B), and 6-, 8-, 9-, and 20-kDa cytochromes (C). (D to F) Effect of oxygen on the fractions containing the 6-, 8-, 9- and 20-kDa cytochromes (D), 6-kDa cytochrome (E), and 10-kDa cytochrome (F).

An earlier investigation has shown that I. dechloratans is cytochrome c oxidase positive with a strictly respiratory type of metabolism (8). We have also observed the UV/visible signature of a cbb₃ oxidase CO complex (11) in a membrane preparation from I. dechloratans (data not shown). I. dechloratans is a facultative anaerobe, and terminal cytochrome c oxidase can utilize the oxygen produced by decomposition of the chlorite produced by chlorate reduction. In order to investigate the electron transfer ability with oxygen as the terminal acceptor in the absence of chlorate, the different cytochrome c fractions were reduced by dithionite and mixed with cell homogenate. Oxygen was added by bubbling oxygen in the cuvette. Figure 4D to F show the spectra of the reduced fractions (Fig. 4D, 6-, 8-, 9-, and 20-kDa cytochromes; Fig. 4E, 6-kDa cytochrome; Fig. 4F, 10-kDa cytochrome) and the effect of adding oxygen in the absence of chlorate. The result shown in Fig. 4D suggests that all components of the fraction containing 6-, 8-, 9-, and 20-kDa cytochromes are oxidized on the addition of oxygen, and the results in Fig. 4E show that the 6-kDa cytochrome is oxidized by oxygen. Spectral changes were, in all cases, complete within 1 minute. The spectral changes produced by the addition of oxygen in Fig. 4D cannot be accounted for by oxidation of the 6-kDa component only. Therefore, the remaining components in this fraction appear to be able to donate electron to oxygen. These results thus suggest that the 6-kDa cytochrome can act as an electron donor both to chlorate reductase and to cytochrome c oxidase, whereas the remaining components in the fraction containing 6-, 8-, 9-, and 20-kDa cytochromes are capable of delivering electrons to cytochrome c oxidase. We were not able to separate the proteins, and therefore, it was not possible to establish the individual roles of the 8-, 9-, and 20-kDa cytochromes. In Fig. 4F, the effect of oxygen on the fraction containing the 10-kDa cytochrome c was investigated. The spectral change produced by the addition of oxygen appears dominated by the 558-nm component in this fraction, and it is difficult from this result to assess the spectral changes due to the 552-nm cytochrome c component.

Figure 4C shows that the fraction containing 6-, 8-, 9-, and 20-kDa c cytochromes was completely reoxidized by the addition of chlorate in the presence of cell homogenate. However, upon chlorate reduction (by the 6-kDa cytochrome), oxygen is produced, which could be used by the terminal cytochrome c oxidase present in the cell homogenate. Reoxidation of the remaining cytochromes in the mixture could be due to oxygen reduction. To investigate their contribution in chlorate reduction only, the experiment shown in Fig. 4C was repeated, but with periplasmic extract as the source of chlorate reductase. In this case, no terminal cytochrome c oxidase was present. The result was an incomplete reoxidation, as shown in Fig. 5. Taken together, these results suggest that the components of this fraction can serve as electron donors for chlorate reductase or the terminal oxidase and that at least one of the components cannot donate electrons directly to chlorate reductase.

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FIG. 5. Effect of chlorate on the fraction containing the 6-, 8-, 9- and 20-kDa cytochromes in the presence of a catalytic amount of chlorate reductase from periplasmic extract, excluding cytochrome c oxidase. The black broken line represents the unreduced state of the fraction as isolated. The black solid line shows the dithionite-reduced state of the fraction. The gray line shows the spectrum obtained after the addition of oxidant. The insert shows the reduced-minus-initial difference spectrum. A, change in absorbance.

In order to test the sensitivity of the soluble c cytochromes for spontaneous air oxidation, periplasmic extract was flushed with nitrogen, reduced by dithionite, and then exposed to air. No spectral changes indicating oxidation were observed. After 10 min, a catalytic amount of cell homogenate was added, and the sample was completely reoxidized.

MS.
The 10-, 9-, 8-, and 6-kDa c cytochromes were excised from SDS-polyacrylamide gels, and peptides were generated by trypsin in-gel digestion. The peptides were analyzed by MALDI-TOF MS. Table 1 summarizes the peaks obtained from the four cytochrome bands.

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TABLE 1. List of peaks from MALDI-TOF MS of in-gel digests of periplasmic c cytochromes from I. dechloratansa

The peptide with an m/z of 1,094.56 (Table 1) from the 6-kDa cytochrome produced a match in the sequence of a type I cytochrome c from Acidovorax avenae subsp. citrulli AAC00-1 (GenBank accession no. YP_969867). The amino acid sequence of the matching peptide is YAGQKDAVDK, as shown in Fig. 6. A majority of the theoretical b- and y-ions could be matched to signals in the MS-MS spectrum of the peptide with an m/z of 1,094.56 (Fig. 7A).

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FIG. 6. Comparison of the sequences obtained for the 6-kDa cytochrome c. Alignment of the cytochrome c sequences from I. dechloratans (I. dech), A. avenae subsp. citrulli (A. ave.sub.citr), and the sequences of three different c cytochromes from D. aromatica. Gaps introduced to maximize alignment are indicated by the dashes. Identical amino acids are indicated by gray shading.

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FIG. 7. Peptide fragment ion spectra of the two peptides with m/z ratios of 1,094.55 (A) and 1,403.76 (B) selected for MS-MS analysis. Mass distances between fragment ion signals matching theoretical y-ions for the two peptides, respectively, are indicated with the corresponding amino acid letter at the top of each spectrum. Additional matching b- and a-ions are also indicated in each spectrum.

In an earlier attempt at de novo sequencing of peptides obtained from c cytochromes, incompletely resolved by SDS-PAGE (BMC Facility, Lund University, Sweden), the sequence KLVGPSYQDVAAR was obtained. A search for a peak at the corresponding m/z of 1,403.76 was therefore conducted in the present data. A candidate was found in the data obtained from the 6-kDa cytochrome (Table 1). MS-MS data from this peak are consistent with the suggested sequence (Fig. 7B). A database search showed that this peptide is similar to the sequence KLVGPAYKDVAAK in three different c cytochromes, class I of the chlorate-reducing Dechloromonas aromatica (GenBank accession no. YP_283918, YP_286520, and YP_283533).

The sequence YAGQKDAVDK (m/z = 1,094.56) described above also shows similarity to the sequence YKGDKGAVDK from one of the D. aromatica c cytochromes (GenBank accession no. YP_283918) and is located next to the KLVGPAYKDVAAK sequence. Figure 6 compares the sequences obtained for the 6-kDa cytochrome c with those of the A. avenae protein and the sequences of the three different c cytochromes of D. aromatica.

The 8- and 9-kDa band proteins produced peptides with a great deal of overlap as seen in Table 1. The observation that a substantial number of the peptide masses are detected in the mass spectra from both bands lends further support to the notion of the 8-kDa band being a degradation product of the 9-kDa protein as discussed above. No proteins were identified in the databases searching with these MS data sets.

Several of the peptides from the 10-kDa band protein were identified as chlorite dismutase, where the residue coverage of the amino acid was 40% (Table 1). The presence of chlorite dismutase at a mass of 10 kDa (intact mass; 25 kDa per subunit) is probably a result of proteolytic degradation. This also explains the shoulder in the reduction peak at 558 nm, due to the heme b in chlorite dismutase (15) in Fig. 4B and F. Our finding that reduction of the 10-kDa fraction produces a spectrum with absorption maximum at 552 nm suggests, however, that a cytochrome c is also present in the fraction.

No other matches were obtained from the database searches. Notably, none of the peptide masses shown in Table 1 are consistent with the protein sequence encoded by the cytochrome c gene located immediately downstream of the chlorate reductase genes in Ideonella dechloratans (GenBank accession no. EU768872). The roles of this protein and its expression conditions remain to be elucidated.

Conclusions.
We have observed that about 55% of the cytochrome c in the periplasmic extract remains reduced after the addition of chlorate, when chlorate acts as the sole terminal electron acceptor (Fig. 2). Also, we have shown that the 6-kDa cytochrome c participates in chlorate reduction, whereas the 10-kDa cytochrome c does not. The 6-kDa band from the gel in Fig. 1 is estimated to constitute about 40% of the low-molecular-mass c cytochromes present in the periplasm. Our results thus show that the soluble 6-kDa cytochrome c is the major, and possibly the sole, mediator for the transfer of electrons between the membrane-bound redox components and the periplasmic chlorate reductase, when cells are anaerobically grown in the presence of chlorate.

The overall reaction, reduction of both chlorate and the produced oxygen (ClO₃^– Cl^– + H₂O), involves transfer of six electrons. The reduction of chlorate to chlorite is a two-electron reaction, whereas reduction of oxygen produced in the subsequent decomposition of chlorite requires four electrons. From the result presented here, we suggest roles for the 6-kDa cytochrome in the electron transport pathway for chlorate reduction in the periplasm of I. dechloratans, as shown in Fig. 8. The 6-kDa cytochrome has a role similar to that of cytochrome c₂ in R. sulfidophilum, but it can also donate electrons to the terminal oxidase, as required for the branched electron flow in chlorate respiration. Additional components capable of donating electrons to the terminal oxidase (8-, 9-, or 20-kDa cytochrome c; Fig. 8) also appear to be present in the periplasm.

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FIG. 8. Proposed electron transfer pathway in I. dechloratans. The mechanism of chlorate reduction includes two steps. After reduction of chlorate, the resulting chlorite is decomposed into chloride ions and molecular oxygen. The produced oxygen can be utilized as a terminal electron acceptor in the absence of an external oxygen supply. The overall reduction of chlorate to chloride involves transfer of six electrons. The reduction of chlorate to chlorite requires two electrons (2e–). The remaining four electrons are consumed when oxygen is reduced to water by the terminal oxidase. Abbreviations: UQ/UQH2, ubiquinone/ubihydroquinone; cyt or cyt., cytochrome.

 
* Corresponding author. Mailing address: Department of Chemistry and Biomedical Sciences, Faculty of Technology and Science, Karlstad University, SE-651 88 Karlstad, Sweden. Phone: 46-54-7001776. Fax: 46-54-7001457. E-mail: thomas.nilsson{at}kau.se

Published ahead of print on 20 February 2009.

Present address: Novozymes Biopharma AB, SE-220 09 Lund, Sweden.

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Applied and Environmental Microbiology, April 2009, p. 2439-2445, Vol. 75, No. 8
0099-2240/09/$08.00+0     doi:10.1128/AEM.01325-08
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