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为硫化物是地球化学硫循环的关键反应，过去已经表征了几种不同的含西罗海姆的亚硫酸盐还原酶。在这里，呼吸亚硫酸盐还原系统（称为Mcc）的调查，使用多血红素细胞色素c亚硫酸盐还原酶MccA作为终端酶的专用电子传递链。在该项目的第一个资助期，研究了由八个基因的mcc簇编码的模式细菌Wolinella succinogenes的Mcc系统，并获得了以下结果。(I)测定了W. succinogenes MccA揭示了MccA同源三聚体中24个血红素C基团的新颖排列和极不寻常的血红素C-亚硫酸铜还原位点。MccA相对于亚硫酸盐和亚硝酸盐还原的酶活性进行了广泛的表征。(II)反应调节剂MccR被发现参与了Mcc系统的上调，以响应亚硫酸盐。(III)非极性突变体缺乏铁硫蛋白MccC或推定的醌醇脱氢酶MccD的表征表明，这两种蛋白质有助于电子传递到MccA。(IV)W.发现缺乏8-MMK的琥珀酸mqnK在亚硫酸盐周转中严重受阻，表明8-甲基甲萘醌（8-MMK）在亚硫酸盐呼吸中起作用。在该项目的过程中，发现MqnK酶负责甲基化产生8-MMK。MqnK是一种新型的自由基S-腺苷甲硫氨酸（SAM）甲基转移酶，可作为产MMK微生物的生物标志物。更新申请介绍了以下主题的工作。(I)它的目的是表征的结构和功能的铜伴侣MccL的背景下，铜掺入到活性位点的MccA。利用分离的MccL进行生理生化实验。(II)详细的作用8-MMK在亚硫酸盐呼吸将被检查。(III)膜蛋白质组学的研究旨在揭示在W.产琥珀酸细胞。(IV)将在转录组水平上仔细检查细胞对亚硫酸盐可用性的反应。总的来说，该项目将有助于彻底了解细菌亚硫酸盐呼吸。

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.