PNNL

Abstract

Sequenced genomes of dissimilatory sulfur-oxidizing and sulfate-reducing bacteria containing genes coding for DsrAB, the enzyme dissimilatory sulfite reductase, inevitably also contain the gene coding for the 12-kDa DsrC protein. DsrC is thought to have a yet unidentified role associated with the activity of DsrAB. Here we report the solution structure of DsrC from the sulfur-oxidizing purple sulfur bacterium Allochromatium vinosum determined with NMR spectroscopy in reducing conditions, and describe the redox behavior of two conserved cysteine residues upon transfer to an oxidizing environment. In reducing conditions, the DsrC structure is disordered in the highly conserved carboxy-terminus. We present multiple lines of evidence that in oxidizing conditions, a strictly conserved cysteine (Cys111) at the penultimate position in the sequence forms an intramolecular disulfide bond with Cys100, which is conserved in DsrC in all organisms with DsrAB. This intramolecularly disulfide-bonded species is the thermodynamically stable form of the protein in oxidizing conditions and forms after rapid formation of the kinetic product, an intermolecular Cys111-Cys111 disulfide-bonded dimer. Treatment of these forms with reducing agent regenerates the monomeric species that was structurally characterized. Furthermore, evidence is provided for strong and specific interaction of DsrC with heterohexameric DsrEFH, a protein encoded in the same operon. Mutation of Cys100 to serine prevented formation of the DsrC species assigned as an intramolecular disulfide in oxidizing conditions, while still allowing formation of the intermolecular Cys111-Cys111 dimer. In the reduced form this mutant protein still interacted with DsrEFH. This was not the case for the Cys111Ser and the Cys100Ser/Cys111Ser mutants, both of which also did not form protein dimers. Our observations highlight the central importance of the carboxy-terminal DsrC cysteine residues and would be consistent with a role as a sulfur-substrate binding/transferring protein as well as with an electron-transfer function via thiol-disulfide interchanges.