Electron Accepting Units of the Diheme Cytochrome c TsdA, a Bifunctional Thiosulfate Dehydrogenase/Tetrathionate Reductase

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 (E_m +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.

Keywords: TsdA; crystal structure; cytochrome c; electron acceptor; enzyme kinetics; heme; protein chemistry; respiratory chain; thiosulfate dehydrogenase.

FIGURE 1.

Redox activity of AvTsdA adsorbed on a mesoporous nanocrystalline SnO₂ electrode. Electronic absorbance was recorded with the electrode poised at +302 mV (black), +152 to −298 mV at 50-mV intervals (gray), and −648 mV (red). All potentials are quoted versus the standard hydrogen electrode (SHE). The arrows indicate increases in absorbance as the electrode potential was lowered. Inset shows the normalized change in absorbance at 418 nm against the applied potential as the enzyme was reduced (closed squares) and re-oxidized (open squares).

FIGURE 2.

UV-visible spectra of TsdB from S. lithotrophicus. As the protein is partly reduced in the “as isolated” state, up to 170 μm ferricyanide were added to record the oxidized spectrum (black line). For full reduction of the protein, sodium dithionite was added (gray line). 100 mm Tris buffer, pH 8.0, with 150 mm NaCl and 2.5 mm desthiobiotin was used, and spectra are normalized to 750 nm. The oxidized spectrum exhibits a 700-nm peak indicating methionine as heme iron ligand. Protein concentration was 6 μm in the overview and 29 μm in the blowup.

FIGURE 3.

Sequence alignment of MpTsdBA and SlTsdB + TsdA as well as TiTsdB + TsdA. Sequence comparison of TsdBA fusion protein of M. purpuratum (MARPU_02550) with the combined sequence of TsdB and TsdA from S. lithotrophicus (Slit_1877 and Slit_1878) and T. intermedia (Tint_1893 and Tint_2892). All signal peptide sequences were removed. Heme-binding motifs are indicated by gray boxes, and putative distal heme ligands are marked by black edging. Strictly conserved residues are marked with asterisks. TsdA sequences of S. lithotrophicus and T. intermedia start after the gap at amino acids 195 and 189, respectively.

FIGURE 4.

Determination of TiTsdB reduction potential by potentiometry with a gold electrode. Potentiometric determination of redox potentials of both TsdB hemes. Applied potential according to normalized values of the α-peak (553 nm) is shown. Reduction of TsdB (black diamonds) and re-oxidation of the protein (gray squares) was measured. 10 μm TsdB in phosphate buffer, pH 5.0, was used. SHE, standard hydrogen electrode.

FIGURE 5.

Analysis of purified SlTsdA and SlTsdB + A by SDS-PAGE. 10–15 μg of SlTsdA and SlTsdB + A obtained after Strep tag affinity chromatography were loaded per lane on a 12.5% gel and stained for presence of heme. In case of SlTsdB + A, both proteins were produced simultaneously in E. coli and purified on the basis of a Strep tag attached to TsdA.

FIGURE 6.

UV-visible spectra of MpTsdBA. As the protein is slightly reduced in the “as isolated” state, 60 μm ferricyanide were added to record the oxidized spectrum (black line). For partial (gray broken line) and full reduction (gray line) of the protein, 0.33 and 5 mm sodium dithionite were added, respectively. 100 mm ammonium acetate buffer, pH 5, with 200 mm NaCl was used, and spectra are normalized to 750 nm. The oxidized spectrum exhibits a 700-nm peak indicating methionine as heme iron ligand and the partially reduced protein exhibits a feature at 630 nm. Protein concentration was 3.3 μm.

FIGURE 7.

X-ray structure of MpTsdBA and heme arrangement. A, overall fold with TsdB N-terminal domain (residues 1–193) depicted in blue and TsdA C-terminal domain (residues 240–516) in red; the two shades in each domain represent the respective sub-domains (1–90 and 91–193 for TsdB and 240–377 and 378–516 for TsdA). The heme prosthetic groups are colored by atom type (orange or yellow for carbon, blue for nitrogen, red for oxygen, and dark red for iron). B, Fe-to-Fe distances; C, closest edge-to-edge distances.

FIGURE 8.

Heme coordination of “as isolated” MpTsdBA (PDB code 5LO9). A, heme 1 is coordinated by His²⁵ and Met⁶⁵. B, heme 2 is coordinated by His¹²⁵ and Met¹⁶⁷. C, heme 3 is ligated to His²⁹¹ but not to Cys³³⁰. The distance of Sγ to the heme iron is 2.9 Å and thus not close enough for direct ligation. Thiosulfate covalently bound to Sγ of Cys³³⁰ is not shown here for clarity. Presence of thiosulfate is illustrated in detail in E and F. D, Heme 4 is ligated by His⁴⁰⁶ and Met⁴⁵⁰. E, heme 3 with Sγ of Cys³³⁰ covalently bound to thiosulfate, displayed in ball and stick, and polder map electron density contoured at 6σ level depicted as a black mesh. F, heme 3 in a similar view as in E but with positively charged residues surrounding the substrate cleft depicted as sticks. Scheme representation is shown in pale gray with heme moieties and coordinating amino acid residues shown as sticks; color code as in Fig. 7 with sulfur atoms in green.

FIGURE 9.

Thiosulfate oxidation catalyzed by AvTsdA and MpTsdBA with HiPIP as electron acceptor. Enzyme assays with AvTsdA were performed in 100 mm ammonium acetate buffer, pH 5, at 30 °C with 8 nm enzyme. Activity measurements with MpTsdBA were performed in 100 mm ammonium acetate buffer, pH 5.2, with 200 mm NaCl at 25 °C and with 3.9 nm enzyme. In both assays 10 μm HiPIP and 40 μm ferricyanide were used. A change in absorbance was measured at 480 nm. v versus [S] plots were fitted to the Hill equation.

FIGURE 10.

Role of periplasmic electron transfer proteins in aerobic respiration or photosynthesis of A. vinosum, M. purpuratum, S. lithotrophicus, and T. intermedia. All organisms contain genes for NuoA-N (Alvin_2418–2430 + Alvin_2412, Marpu_04365–04430, Slit_1070–1083, and Tint_2255–2268), the cytochrome bc₁ complex (Alvin_0068–0070, Marpu_01465–01475, Slit_0130–0132, and Tint_2192–2194), and cbb₃ oxidase (Alvin_0781–0784, Marpu_02795–02810, Slit_0411–0414, and Tint_1070–1073). Moreover, A. vinosum and M. purpuratum can gain energy by photosynthetic growth. HiPIP can transfer electrons to the photosynthetic reaction center (23, 24) as well as to cbb₃ oxidase (29, 30). Cytochrome c₄ also is known to transfer electrons to the photosynthetic reaction center (22) as well as to cbb₃ oxidase (32–34) in some bacteria. For S. lithotrophicus, it is assumed that MtoD (Slit_2498) can transfer electrons to cbb₃ oxidase and the cytochrome bc₁ complex (31).