Abstract
The enzymes of the thiosulfate dehydrogenase (TsdA) family are wide-spread diheme c-type cytochromes. Here, redox carriers were studied mediating the flow of electrons arising from thiosulfate oxidation into respiratory or photosynthetic electron chains. In a number of organisms, including Thiomonas intermedia and Sideroxydans lithotrophicus, the tsdA gene is immediately preceded by tsdB encoding for another diheme cytochrome. Spectrophotometric experiments in combination with enzymatic assays in solution showed that TsdB acts as an effective electron acceptor of TsdA in vitro when TsdA and TsdB originate from the same source organism. Although TsdA covers a range from -300 to +150 mV, TsdB is redox active between -100 and +300 mV, thus enabling electron transfer between these hemoproteins. The three-dimensional structure of the TsdB-TsdA fusion protein from the purple sulfur bacterium Marichromatium purpuratum was solved by X-ray crystallography to 2.75 Å resolution providing insights into internal electron transfer. In the oxidized state, this tetraheme cytochrome c contains three hemes with axial His/Met ligation, whereas heme 3 exhibits the His/Cys coordination typical for TsdA active sites. Interestingly, thiosulfate is covalently bound to Cys330 on heme 3. In several bacteria, including Allochromatium vinosum, TsdB is not present, precluding a general and essential role for electron flow. Both AvTsdA and the MpTsdBA fusion react efficiently in vitro with high potential iron-sulfur protein from A. vinosum (Em +350 mV). High potential iron-sulfur protein not only acts as direct electron donor to the reaction center in anoxygenic phototrophs but can also be involved in aerobic respiratory chains.
Highlights
Between ؊100 and ؉300 mV, enabling electron transfer between these hemoproteins
The enzyme from the sulfur oxidizer A. vinosum is strongly adapted to catalyzing thiosulfate oxidation [2], thiosulfate dehydrogenase (TsdA) from Campylobacter jejuni acts primarily as a tetrathionate reductase and enables the organism to use tetrathionate as an alternative electron acceptor for anaerobic respiration [8]
Isms, including Thiomonas intermedia, Sideroxydans lithotrophicus, and Pseudomonas stutzeri, tsdA is immediately preceded by a gene encoding another diheme cytochrome, TsdB [1]
Summary
Characterization of TsdA and TsdB—UV-visible electronic absorbance spectroscopy, X-ray diffraction, and activity assays have revealed a number of characteristic features of AvTsdA [1, 2, 12]. The electrochemical window in which the hemes are redox active remained unknown. To gain insight into this property, we mapped out the redox activity of AvTsdA adsorbed as an electroactive film on optically transparent mesoporous nanocrystalline SnO2 electrodes. The spectrum of the enzyme-coated electrode equilibrated at ϩ302 mV contained features typical of ferric c-type hemes superimposed on a small contribution from light scattering by the electrode material (Fig. 1). The Soret maximum at 406 nm and broad lower intensity features in the ␣-region are typical of those displayed by solutions of oxidized AvTsdA [1]. When the electrode potential was lowered to Ϫ648 mV in 50-mV steps with a spectrum recorded after a 60-s pause at each desired potential
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