Bacterial sulfite respiration: structure, function and biogenesis of the Mcc system

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Despite its reactivity and toxicity to living cells, sulfite is readily converted by microorganisms. Assimilatory and dissimilatory sulfite reduction to sulfide are key reactions of the biogeochemical sulfur cycle and several distinct sirohaem-containing sulfite reducing enzymes have been characterized in the past. Here, a respiratory sulfite reduction system (called Mcc) is investigated that uses the multihaem cytochrome c sulfite reductase MccA as terminal enzyme of a dedicated electron transport chain. In the first funding period of this project the Mcc system of the model bacterium Wolinella succinogenes, encoded by the mcc cluster of eight genes, has been investigated and the following results have been obtained. (I) Determination of the high-resolution crystal structure of W. succinogenes MccA revealed a novel arrangement of 24 haem c groups in the MccA homotrimer and a highly unusual haem c-copper sulfite reduction site. The enzymatic activity of MccA with respect to sulfite and nitrite reduction was extensively characterized. (II) The response regulator MccR was found to be involved in the upregulation of the Mcc system in response to sulfite. (III) The characterization of non-polar mutants lacking either the iron-sulfur protein MccC or the putative quinol dehydrogenase MccD indicated that these two proteins contribute to electron transport to MccA. (IV) Cells of W. succinogenes mqnK lacking 8-MMK were found to be severely hampered in sulfite turnover indicating that 8-methylmenaquinone (8-MMK) plays a role in sulfite respiration. In the course of the project the enzyme MqnK was found to be responsible for menaquinone methylation yielding 8-MMK. MqnK turned out to be a novel radical S-adenosylmethionine (SAM) methyltransferase applicable as a biomarker for MMK-producing microorganisms. The renewal application describes work on the following topics. (I) It is aimed to characterize the structure and function of the copper chaperone MccL in the context of copper incorporation into the active site of MccA. Physiological and biochemical experiments using isolated MccL are proposed. (II) The detailed role of 8-MMK in sulfite respiration will be examined. (III) Membrane proteomics studies are intended to reveal the architecture of the sulfite-reducing respiratory Mcc system in the membrane of W. succinogenes cells. (IV) The cellular response to sulfite availability will be scrutinized at the transcriptomic level. Overall, the project will contribute to a thorough understanding of bacterial sulfite respiration.