Thiosulfate dehydrogenase: an unusual acidophilic c-type cytochrome
Final Report Abstract
TsdA enzymes were identified as a phylogenetically widespread family of periplasmic c-type diheme cytochromes which catalyze both thiosulfate oxidation and tetrathionate reduction. The reaction directionality varies between enzymes isolated from different bacteria and is in line with their different physiological functions: TsdA enables the purple sulfur bacteria Allochromatium vinosum (Av) and Marichromatium purpuratum (Mp) to use thiosulfate as an electron donor for photosynthesis or respiration whereas the enzyme from the human gut pathogen Campylobacter jejuni (Cj) allows the organism to use tetrathionate as a terminal electron acceptor. The bifunctionality of the TsdA enzyme allowed experimental determination of the reduction potential of the tetrathionate/thiosulfate couple, ETT/TS. This was one of the most important results of the project as previous calculations from the relevant thermodynamic data had yielded conflicting results. An ETT/TS value of +198 mV was obtained which is much more positive than the value of +24 mV widely cited in the field of microbial bioenergetics. As a consequence, more free energy is available to be harnessed during the respiratory reduction of tetrathionate to thiosulfate than was previously recognized and dissimilatory tetrathionate reduction is likely to be more prevalent in tetrathionate-containing environments such as the human gut or marine sediments than presently thought. Structure, kinetics as well as electrochemical and biophysical properties were characterized in detail for wildtype and variant TsdA enzymes from A. vinosum, M. purpuratum and C. jejuni. The TsdA active site hemes all show an unusual His/Cys ligation which is of special importance in sulfurbased energy metabolism. In contrast, the ligand constellation of the electron transfer hemes (Heme 2) differs depending on the source organism. In the case of AvTsdA, methionine ligates Heme 2 iron only in the reduced state, while in CjTsdA this residue also acts as a Heme 2 iron ligand in the oxidized state. In the case of CjTsdA, the redox activity of the negative potential heme, Heme 1, reaches down to -650 mV and that of the positive potential heme, Heme 2, up to +300 mV. Moreover, a strong cooperativity between the two TsdA hemes was observed. In vitro as well as in vivo experiments with CjTsdA revealed that structural differences in the immediate environment of Heme 2 contribute to defining the reaction directionality. The TsdA reaction cycle was elucidated and proven to involve covalent linkage between thiosulfate and the active site cysteine. As a consequence, a thiol-disulfide exchange is the second important step of the TsdA reaction mechanism. The covalent linkage of a sulfur species to the active site cysteine constitutes the rate-defining step within the TsdA reaction cycle. A positive shift in the Heme 1 reduction potential occurs during formation of the covalent bond facilitating electron transfer during the reaction cycle. The substrate mimic sulfite was identified as a competitive inhibitor. The diheme cytochrome c TsdB was identified as an effective electron acceptor of TsdA in vitro when TsdA and TsdB originate from the same source organism. For AvTsdA and the MpTsdBA fusion protein the high potential iron-sulfur protein HiPIP was identified as a suitable electron acceptor. In contrast to all other tsdA containing organisms, the tsdA gene from Wolinella succinogenes is preceded by tsdC. TsdC is a novel and unique membrane attached lipoprotein that directly interacts with TsdA and does not contain prosthetic groups. Similar to CjTsdA, TsdA from W. succinogenes predominantly acts as a tetrathionate reductase. In contrast to the situation in C. jejuni, a membrane attachment of the tetrathionate reductase by the lipoprotein TsdC seems to be indispensable for tetrathionate reduction in W. succinogenes.
Publications
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(2012) Thiosulfate dehydrogenase: a widespread unusual acidophilic c-type cytochrome. Environ. Microbiol. 14, 2673-2688
Denkmann, K., Grein, F., Zigann, R., Siemen, A., Bergmann, J., van Helmont, S., Nicolai, A., Pereira, C. & Dahl, C.
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(2013) Tetrathionate stimulated growth of Campylobacter jejuni identifies TsdA as a new type of bi-functional tetrathionate reductase that is widely distributed in bacteria. Mol. Microbiol. 88, 173-188
Liu, W.-Y., Denkmann, K., Kosciow, K., Dahl, C. & Kelly, D.J.
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(2014) Production, crystallization and preliminary crystallographic analysis of Allochromatium vinosum TsdA, an unusual acidophilic c-type cytochrome. Acta Cryst. F70, 1424-1427
Brito, J., Gutierres, A., Denkmann, K., Dahl, C. & Archer, M.
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(2015) Catalytic protein film electrochemistry provides a direct measure of the tetrathionate/thiosulfate reduction potential. J. Am. Chem. Soc. 137, 13232-13235
Kurth, J., Dahl, C. & Butt, J.
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(2015) Thiosulfate dehydrogenase (TsdA) from Allochromatium vinosum: Structural and functional insights into thiosulfate oxidation. J. Biol. Chem. 290, 9222-9238
Brito, J., Denkmann, K., Pereira, I. A. C., Archer, M. & Dahl, C.
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(2016) Electron accepting units of the diheme cytochrome c TsdA, a bifunctional thiosulfate dehydrogenase/ tetrathionate reductase. J. Biol. Chem. 291, 24804-24818
Kurth, J.M., Brito, J.A., Reuter, J., Flegler, A., Koch, T., Franke, T., Klein, E.M., Rowe, S.F. Butt, J.N., Denkmann, K., Pereira, I.A., Archer, M. & Dahl, C.
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(2016) Influence of heme environment on the catalytic properties of tetrathionate reductase (TsdA) from Campylobacter jejuni. Biosci. Rep. e00422
Kurth, J.M., Butt, J.N., Kelly, D.J. & Dahl, C.
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(2017) Ein altes Paar in neuem Glanz: Thiosulfat und Tetrathionat. BIOspektrum 23, 25-27
Kurth, J.M. & Dahl, C.
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(2017) Sulfur metabolism in phototrophic bacteria. In Modern topics in the phototrophic prokaryotes. Metabolism, bioenergetics and omics (Hallenbeck, P. C., ed.) Springer, Cham, 27-66
Dahl, C.
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(2017) TsdC, a unique lipoprotein from Wolinella succinogenes that enhances tetrathionate reductase activity of TsdA. FEMS Microbiol. Lett. 364, fnx003
Kurth, J.M., Schuster, A., Seel, W., Herresthal, S., Simon, J. & Dahl, C.
