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

尽管亚硫酸盐对活细胞具有反应性和毒性，但它很容易被微生物转化。同化和异化亚硫酸盐还原成硫化物是生物地球化学硫循环的关键反应，过去已经鉴定了几种不同的含siro血红的亚硫酸盐还原酶。本文研究了一种呼吸亚硫酸盐还原系统（Mcc），该系统使用多血红素细胞色素c亚硫酸盐还原酶MccA作为专用电子传递链的末端酶。在本项目第一期资助期内，对由8个Mcc基因簇编码的模式细菌琥珀酸Wolinella succinogenes的Mcc系统进行了研究，并获得了以下结果：(I)对W. succinogenes MccA的高分辨率晶体结构的测定揭示了MccA同源三聚体中24个血红素c基团的新排列和一个非常不寻常的血红素c-亚硫酸铜还原位点。对MccA在亚硫酸盐和亚硝酸盐还原方面的酶活性进行了广泛的表征。（II）响应调节因子mcr被发现参与Mcc系统对亚硫酸盐的响应上调。（III）缺乏铁硫蛋白MccC或假定的喹啉脱氢酶MccD的非极性突变体的特征表明，这两种蛋白质有助于MccA的电子传递。（IV）研究发现，缺乏8-MMK的W. succinogenes mqnK细胞的亚硫酸盐转换受到严重阻碍，这表明8-甲基萘醌（8-MMK）在亚硫酸盐呼吸中起作用。在项目过程中，发现MqnK酶负责甲基化，产生8-MMK。结果表明，MqnK是一种新型的自由基s -腺苷蛋氨酸（SAM）甲基转移酶，可作为产生mmk的微生物的生物标志物。续订申请描述了以下主题的工作。(1)在铜掺入MccA活性位点的情况下，对铜伴侣蛋白MccL的结构和功能进行表征。提出了利用分离的mcl进行生理生化实验。（II）将研究8-MMK在亚硫酸盐呼吸中的详细作用。（III）膜蛋白质组学研究旨在揭示W. succinogenes细胞膜中亚硫酸盐还原呼吸Mcc系统的结构。（IV）细胞对亚硫酸盐可用性的反应将在转录组水平上进行仔细研究。总的来说，该项目将有助于彻底了解细菌亚硫酸盐呼吸作用。

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.