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Applied and Environmental Microbiology, November 2008, p. 6808-6810, Vol. 74, No. 21
0099-2240/08/$08.00+0     doi:10.1128/AEM.00787-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Reconstitution of Iron Oxidase from Sulfur-Grown Acidithiobacillus ferrooxidans{triangledown} ,{dagger}

Taher M. Taha,1* Tadayoshi Kanao,1 Fumiaki Takeuchi,2 and Tsuyoshi Sugio1

Division of Bioscience, Graduate School of Natural Science and Technology, Okayama University, 1-1-3 Tsushima-naka, Okayama 700-8530, Japan,1 Environmental Management Center, Okayama University, 1-1-3 Tsushima-naka, Okayama 700-8530, Japan2

Received 7 April 2008/ Accepted 30 August 2008


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ABSTRACT
 
The iron oxidation system from sulfur-grown Acidithiobacillus ferrooxidans ATCC 23270 cells was reconstituted in vitro. Purified rusticyanin, cytochrome c, and aa3-type cytochrome oxidase were essential for reconstitution. The iron-oxidizing activity of the reconstituted system was 3.3-fold higher than that of the cell extract from which these components were purified.


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INTRODUCTION
 
Acidithiobacillus ferrooxidans has enzyme systems that can oxidize not only ferrous iron but also reduced sulfur compounds. One big controversy in the study of energy generation in A. ferrooxidans is whether the iron oxidation enzyme system, containing rusticyanin, cytochrome c, and aa3-type cytochrome c oxidase, is involved in sulfur oxidation. Sugio et al. (9, 10) proposed the involvement of an iron oxidation enzyme system in sulfur oxidation of iron-grown A. ferrooxidans cells by the oxidation of ferrous iron produced from the reduction of ferric iron by sulfide:ferric ion oxidoreductase (H2S + 6Fe3+ + 3H2O -> H2SO3 + 6Fe2+ + 6H+) and sulfite:ferric ion oxidoreductase (H2SO3 + 2Fe3+ + H2O -> H2SO4 + 2Fe2+ + 2H+). In contrast, some other researchers suggest the involvement of sulfide:quinone oxidoreductase, from A. ferrooxidans NASF-1 (15), and thiosulfate:quinone oxidoreductase, from A. ferrooxidans ATCC 19859 (2), in electron transport from sulfide and thiosulfate, respectively, to the quinone pool, and then the electrons may be transported directly from the quinone pool to bd quinol oxidase or to ba3 cytochrome c oxidase via the bc1 II complex and c4 cytochrome (2). In this model, bd quinol oxidase and ba3 cytochrome oxidase, but not aa3-type cytochrome c oxidase, are working as the terminal oxidases in sulfur oxidation.

In this work we partially purified rusticyanin and cytochrome c as well as aa3-type cytochrome c oxidase from sulfur-grown A. ferrooxidans ATCC 23270 cells and show for the first time that ferrous iron oxidation activity, higher than that observed in the cell extract, could be detected with the combination of all of these three components.

In brief (detailed methods can be found in the supplemental material), A. ferrooxidans ATCC 23270 was grown in sulfur medium in the absence of iron for 4 days and then harvested as described by Taha et al. (12). Crude cell extract was prepared by solubilization of the resting cells in 0.1 M β-alanine-SO42– buffer (pH 2) containing 2% 1-O-n-octyl-β-D-glucopyranoside (OGL) and 1 M Na2SO4 for 60 min. Rusticyanin, aa3-type cytochrome c oxidase, and cytochrome c were purified from the solubilized crude extract using ammonium sulfate precipitation and Phenyl-650 M and Superdex 75 column chromatographies. Ferrous iron oxidation activity was measured by the o-phenanthroline method (8, 11). Protein concentration was measured by the method of Lowry et al. (6). The spectra of cytochrome c, aa3-type cytochrome oxidase, and rusticyanin were measured with a Shimadzu UV-1700 spectrophotometer. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) was done by the method of Laemmli (5). After electrophoresis, gels were stained by Coomassie blue or the protein bands on the gel were transferred to a polyvinylidene difluoride membrane by following the technique described by Ausubel et al. (1). The intended bands were excised, dried at room temperature, and kept at –20°C until sequencing by an Applied Biosystems 491 protein sequencer.

The genes involved in ferrous iron oxidation by A. ferrooxidans cells form the rus operon which encodes two cytochromes c, aa3-type cytochrome c oxidase, and rusticyanin (17). Therefore, we tried to purify these components from sulfur-grown A. ferrooxidans ATCC 23270 cells. SDS-PAGE of the partially purified components (Fig. 1) showed only one band at 18.0 kDa in the rusticyanin fraction and three bands with molecular sizes of 57.2 ({alpha}-band), 25.1 (β-band), and 20.1 ({gamma}-band) kDa in aa3-type cytochrome c oxidase. Two bands at 47.0 and 25.0 kDa were detected in the cytochrome c fraction, suggesting the existence of the two cytochromes c encoded by rus operon genes Cyc2 and Cyc1, respectively (16, 17). Spectral analysis of partially purified rusticyanin, cytochrome c, and aa3-type cytochrome c oxidase fractions were in complete accordance with the literature (3, 4, 7, 11) (data not shown). N-terminal amino acid sequencing of 10 residues of the 18-kDa band of the rusticyanin fraction, the 20.1-kDa band of the aa3-type cytochrome c oxidase fraction ({gamma}-band), and the 25.0-kDa and 47.0-kDa bands of the cytochrome c fraction were carried out. The obtained sequences were GTLDTTWKEA, AAKKGMTTVL, AVGSADAPAP, and LPSFARQTGW, respectively. Searching for these sequences using the websites of The Institute for Genomic Research (http://tigrblast.tigr.org/cmr-blast/) and the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/projects/gorf/) and the GENETYX software showed that the above sequences are the same as those of rusticyanin, the {gamma}-band of aa3-type cytochrome c oxidase, Cyc1 cytochrome, and Cyc2 cytochrome of A. ferrooxidans ATCC 23270, respectively.


Figure 1
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  		FIG. 1. Coomassie blue-stained SDS-PAGE of partially purified rusticyanin (left), aa3-type cytochrome c oxidase (middle), and cytochrome c (right) from sulfur-grown A. ferrooxidans ATCC 23270. The molecular masses of the protein size marker and the partially purified rusticyanin, aa3-type cytochrome oxidase, and cytochrome c are expressed in kDa. Five micrograms from each fraction was applied to the gel. In the case of rusticyanin, 15% polyacrylamide gel was used, while in the case of aa3-type cytochrome c oxidase and cytochrome c, 12.5% polyacrylamide gel was used.

The abilities of these partially purified proteins from sulfur-grown A. ferrooxidans ATCC 23270 cells to oxidize ferrous iron were studied either separately or in combinations. The combination of these three fractions showed higher activity (3.9 nmol Fe2+ oxidized mg protein–1 min–1) than that of the cell extract, from which these components were purified (1.2 nmol Fe2+ oxidized mg protein–1 min–1) (Table 1). These results indicate that rusticyanin, two cytochromes c (Cyc1 and Cyc2), and aa3-type cytochrome c oxidase are the essential components of the iron oxidation system in the sulfur-grown cells. The low enrichment factor (3.3-fold) may be due to the very complicated nature of the iron oxidation enzyme system of A. ferrooxidans because (i) it is composed of at least four protein components, rusticyanin, two cytochromes c (Cyc1 and Cyc2), and aa3-type cytochrome c oxidase, and (ii) these components are localized in different cell loci (outer membrane, periplasmic space, and inner membrane) with proper configurations in the intact cells of A. ferrooxidans. We observed that a high iron-oxidizing activity of the intact cells decreased dramatically when a cell-free iron oxidation system was prepared. These results suggest that the functional localizations and/or configurations of the four components cannot be achieved in an in vitro assay system, neither with the crude cell extract nor with the purified reconstituted system. This explanation is strongly supported by the observation that the iron oxidation activity of the resting cells of A. ferrooxidans ATCC 23270 dramatically decreased compared to that of strain TI-1, a moderately thermophilic iron-oxidizing bacterium with a simple iron oxidation system composing only aa3-type cytochrome c oxidase but not rusticyanin and cytochrome c (13, 14). The direct extraction method, used in this study, gave approximately one-five hundredth of the iron oxidation activity of not only sulfur-grown but also iron-grown ATCC 23270 cells. Although an improvement should be done to prepare a more-active cell-free system, we are thinking that the dramatic decrease of iron-oxidizing activity after disrupting the intact cells is an intrinsic property of the elegant and complicated iron oxidation system of A. ferrooxidans. The possibility that our reconstituted system might miss some important components for iron oxidation seems to be very weak, because in the absence of any of the four essential components, activity could not be detected (Table 1).


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  		TABLE 1. Reconstitution of ferrous iron oxidation activity from the partially purified rusticyanin, cytochrome c, and aa3-type cytochrome c oxidase from sulfur-grown A. ferrooxidans ATCC 23270 cells separately or in combinationa

To date there is no report of the reconstitution of the ferrous iron oxidation system from sulfur-grown A. ferrooxidans ATCC 23270 cells. In this report, all the components needed for iron oxidation by iron-grown A. ferrooxidans ATCC 23270 cells, such as rusticyanin, cytochromes c, and aa3-type cytochrome c oxidase, were purified from sulfur-grown A. ferrooxidans ATCC 23270 cells. We showed that iron oxidation activity approximately 3.3-fold higher than that observed in the cell extract was detected when all three fractions purified from sulfur-grown ATCC 23270 cells were added together to the reaction mixture. However, the addition of these fractions separately or in combinations of two did not show any iron oxidation activity. These results indicate that the iron oxidation enzyme system of sulfur-grown A, ferrooxidans ATCC 23270 cells was reconstituted for the first time with rusticyanin, two cytochromes c (Cyc1 and Cyc2), and aa3-type cytochrome c oxidase from sulfur-grown cells.

To clarify the sulfur oxidation mechanism in an industrially important bacterium more precisely, we are now studying whether both the cytochrome c and aa3-type cytochrome c oxidase purified from sulfur-grown A. ferrooxidans ATCC 23270 cells can be reduced enzymatically in the presence of reduced sulfur compounds.


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ACKNOWLEDGMENTS
 
Taher M. Taha is supported by the Egyptian Ministry of Higher Education through scholarship no. 1/5/42.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Bioscience, Graduate School of Natural Science and Technology, Okayama University, 1-1-3 Tsushima-naka, Okayama 700-8530, Japan. Phone and fax: 81-86-251-8306. E-mail: tahery1{at}yahoo.com Back

{triangledown} Published ahead of print on 12 September 2008. Back

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


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Applied and Environmental Microbiology, November 2008, p. 6808-6810, Vol. 74, No. 21
0099-2240/08/$08.00+0     doi:10.1128/AEM.00787-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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