Evaluating the role of DsrC as a key protein in dissimilatory sulfur metabolism
EMSL Project ID
35205
Abstract
The dissimilatory reduction of sulfur compounds is one of the earliest energy metabolisms detected on Earth, at ~3.5 billion years ago. Sulfate reducing bacteria (SRB) are prominent among sulfur-metabolising prokaryotes due to their ubiquitous presence in the environment. SRB are major contributors to the biological carbon and sulfur cycles and their activities have important environmental and economic consequences (9), such as bioremediation of metal-contaminated soils. A key enzyme in sulfur-based energy metabolism is the dissimilatory sulfite reductase (dSiR), which is composed of two subunits, DsrA and DsrB, in an α2β2 arrangement. All organisms containing a dSiR include also the genes for the DsrC protein and the DsrMKJOP complex. DsrC is one of the most highly expressed proteins in SRB, pointing to a very important role in cellular metabolism. In sulfate reducers of the genus Desulfovibrio the dSiR forms a stable complex with DsrC, and we recently solved the crystal structure of this complex. Homologues of DsrC, like TusE, are also present in organisms that do not contain dSiRs such as E.coli where it is involved in sulfur-transfer reactions. The functional part of DsrC seems to be a highly conserved, flexible C-terminal arm, with several strictly conserved residues including two cysteines. There is evidence for the possible involvement of a disulfide bridge between these cysteines as a redox-active center in the sulfite reduction pathway. In the DsrABC complex the C-terminal arm of DsrC reaches the catalytic siroheme with Cys104 sulfur being positioned right next to the substrate binding side. A mechanism was proposed for the reduction of sulfite involving a persulfide of DsrC Cys104 as intermediate. The structure of reduced DsrC contains a region of approximately 50 residues that closely resembles the helix-turn-helix motif of transcriptional regulatory proteins. Preliminary gel shift assays suggest DsrC from a sulfur oxidizing bacterium does interact with DNA upstream from the dsr operon. The finding of a DsrC-DNA interaction suggests a role in transcriptional regulation for DsrC, making this a novel class of regulatory proteins that is directly involved in a fundamental aspect of metabolism.This project aims to elucidate the role played by DsrC in dissimilatory sulfate and sulfite reduction. For this we will prepare DsrC in different oxidation states (reduced and oxidised monomer, dimer), which will be characterized by FTICR-MS and NMR, and tested as possible substrates for the DsrMKJOP complex. We will also investigate conditions to form a DsrC persulfide, and study its reactivity. The redox status of DsrC in the cell will be tested by growth in different conditions by mass spectrometry, and also in vivo NMR of D. vulgaris grown on stable isotope (13C, 15N) labeled media. Mass spectrometry will be used to assess the stoichiometry of DsrC:DsrAB in different forms of the complex. Using NMR we will study the DsrC-DNA interactions. The assigned chemical shifts of DvH DsrC will allow detection of DNA binding and assessment of the binding surface on DsrC using chemical shift perturbation mapping.
This work will be a decisive contribution to understanding a fundamental biological process that has economic, environmental and health repercussions.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2009-10-08
End Date
2012-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Venceslau SS, JR Cort, ES Baker, RK Chu, EW Robinson, C Dahl, LM saraiva, and A pereira. 2013. "Redox states of Desulfovibrio vulgaris DsrC, a key protein in dissimilatory sulfite reduction." Biochemical and Biophysical Research Communications 441:731-736. doi:10.1016/j.bbrc.2013.10.116