MALDI-TOF spectra of persulfurated DsrC proteins.

<p>30 µM of unmodified DsrC <b>(A)</b> and DsrC mutant proteins carrying a Cys-Ser mutation in DsrC-Cys100 (<b>B</b>) or DsrC-Cys111 (<b>C</b>) were incubated with 2 µM IscS and 2 mM cysteine or with 2 mM sulfide. Note that for the unmodified DsrC results are shown for the double charged molecule.</p

MALDI-TOF spectra of persulfurated DsrEFH proteins.

<p>30 µM of unmodified DsrEFH and protein carrying a Cys-Ser mutation in DsrE were incubated with 2 µM IscS and 2 mM cysteine or 2 mM sulfide. Binding of sulfur atoms is indicated by an additional mass of 32 Da for singly charged molecules and 16 Da for double charged molecules. Note that results are shown for double charged proteins. <b>(A)</b> DsrE.(<b>B</b>) DsrF. (<b>C</b>) DsrH. (<b>D</b>) DsrE₇₈.</p

Formation of stable protein complexes between DsrC and DsrEFH analysed with electrophoretic methods.

<p>(<b>A</b>) For the interaction, 200 pmol of DsrEFH and 400 pmol of DsrC were incubated for 30 minutes at 30°C. The protein mixtures were then applied to a native polyacrylamide gel (7.5%). The additional bands that indicate DsrEFH/DsrC complexes are marked by * and **. All bands were cut out of the gel and the pieces were applied to SDS-PAGE (15%). (<b>A</b>) Proteins in native gel (lanes 1–3): lane 1 DsrEFH, lane 2 DsrC, lane 3 DsrEFH pre-incubated with DsrC; (<b>B</b>) SDS-PAGE (lanes 4–7): lane 4 DsrEFH, lane 5 DsrC, lane 6 DsrEFH and DsrC (lower migrating band), lane 7 DsrEFH and DsrC (upper migrating band). Molecular weight (MW) of marker proteins is given in kDa. (<b>C</b>) Proteins in Blue-native PAGE (10–15%): lane 1 DsrEFH, lane 2 to 4 DsrEFH pre-incubated with DsrC in 1∶2 ratio but with increasing amounts. (<b>D</b>) DLS measurements of DsrEFH (black line) and DsrC (dashed line) in solution, and when both proteins were pre-incubated together at 30°C (grey line).</p

MALDI-TOF spectrum of DsrC.

<p>30 µM DsrC was incubated with 30 µM persulfurated DsrEFH for 1 hour at 30°C. The transfer of up to three sulfur atoms from DsrEFH to DsrC is documented by mass increase in steps of 32 Da.</p

Cytoplasmic Sulfurtransferases in the Purple Sulfur Bacterium <em>Allochromatium vinosum:</em> Evidence for Sulfur Transfer from DsrEFH to DsrC

<div><p>While the importance of sulfur transfer reactions is well established for a number of biosynthetic pathways, evidence has only started to emerge that sulfurtransferases may also be major players in sulfur-based microbial energy metabolism. Among the first organisms studied in this regard is the phototrophic purple sulfur bacterium <em>Allochromatium vinosum</em>. During the oxidation of reduced sulfur species to sulfate this Gammaproteobacterium accumulates sulfur globules. Low molecular weight organic persulfides have been proposed as carrier molecules transferring sulfur from the periplasmic sulfur globules into the cytoplasm where it is further oxidized via the “Dsr” (<b><u>d</u></b>issimilatory <b><u>s</u></b>ulfite <b><u>r</u></b>eductase) proteins. We have suggested earlier that the heterohexameric protein DsrEFH is the direct or indirect acceptor for persulfidic sulfur imported into the cytoplasm. This proposal originated from the structural similarity of DsrEFH with the established sulfurtransferase TusBCD from <em>E. coli</em>. As part of a system for tRNA modification TusBCD transfers sulfur to TusE, a homolog of another crucial component of the <em>A. vinosum</em> Dsr system, namely DsrC. Here we show that neither DsrEFH nor DsrC have the ability to mobilize sulfane sulfur directly from low molecular weight thiols like thiosulfate or glutathione persulfide. However, we demonstrate that DsrEFH binds sulfur specifically to the conserved cysteine residue DsrE-Cys78 <em>in vitro</em>. Sulfur atoms bound to cysteines in DsrH and DsrF were not detected. DsrC was exclusively persulfurated at DsrC-Cys111 in the penultimate position of the protein. Most importantly, we show that persulfurated DsrEFH indeed serves as an effective sulfur donor for DsrC <em>in vitro.</em> The active site cysteines Cys78 of DsrE and Cys20 of DsrH furthermore proved to be essential for sulfur oxidation <em>in vivo</em> supporting the notion that DsrEFH and DsrC are part of a sulfur relay system that transfers sulfur from a persulfurated carrier molecule to the dissimilatory sulfite reductase DsrAB.</p> </div