Structure and function of a bacterial ion (Na+)-translocating ferredoxin: NAD+-oxidoreductase (Rnf)

厌氧产乙酸菌伍迪醋酸菌生活在热力学生命极限。在自养生长过程中，底物水平磷酸化的净ATP合成为零，但假设一个非常简单的呼吸链，由膜结合、Na+转位的铁氧还蛋白：NAD氧化还原酶和Na+-F1FO ATP合成酶组成，以合成少量的ATP(形成0.3mol/mol醋酸酯)。我们早期的研究表明，铁氧还蛋白：NAD氧化还原酶是由rnf基因编码的，这类基因早在20世纪80年代就已经被其他人提出，编码一种与Na+转运型NADH：苯醌氧化还原酶相似的呼吸酶。Rnf基因广泛存在于不同的细菌系统发育类群和一些古生菌中。然而，对RNF复合体的功能和生理作用的详细了解还很少。在上一次资助期间，我们更详细地描述了RNF在木兰中的生理作用，阐述了该活性的生物化学和生物能量学，并确定了RNF复合体及其亚单位的性质。利用细菌和考古子进行的缺失分析提供了铁氧还蛋白：NAD氧化还原酶活性确实是由rnf基因编码的证据。我们为该复合体建立了可靠的酶分析方法，明确地证明了该复合体对Na+的依赖性，并丰富了该复合体中的Na+-RNF复合体以及Na+-ATP合成酶。一个非常重要的观察结果是，二环己基碳二亚胺(DCD)抑制了铁氧还蛋白依赖的NAD还原，这种抑制可以通过预先与Na+孵育来阻止。此外，我们成功地在A.wodii中同源生产了蛋白质，这将为RNF复合体的结构功能分析打开大门。我们将跟进在第一个资助期内获得的数据，并将追求两个主要目标。首先，上面描述的Na+-RNF和Na+-F1FO ATP合成酶的制备提供了独特的机会来验证RNF活性与离子转运和膜电位的产生耦合从而驱动ATP合成的假说。为此，制剂中存在的RNF和ATP合成酶将被共重组为脂质体，并进行生物化学和生物能量分析。其次，RNF复合体的特征将集中在参与离子结合的亚基和残基上，利用DCCD和Na+的竞争。DCCD法将被用作确定Na+结合部位的工具(S)。我们最近还建立了一种在木霉中生产蛋白质的程序。这将使我们能够通过突变分析来验证DCCD结合位点对Na+转运的重要性，并允许确定Na+结合口袋的其他残基，甚至额外的Na+结合口袋。总而言之，我们将对一种新的、迄今尚未研究过的呼吸酶的功能有了详细的了解。

The anaerobic acetogenic bacterium Acetobacterium woodii lives at the thermodynamic limit of life. During autotrophic growth, net ATP synthesis by substrate level phosphorylation is zero, but a very simple respiratory chain consisting of a membrane-bound, Na+-translocating ferredoxin:NAD oxidoreductase and a Na+-F1FO ATP synthase is hypothesized to synthesize a little ATP in addition (0.3 mol/mol acetate formed). Our own earlier studies suggested that the ferredoxin:NAD oxidoreductase is encoded by the rnf genes, a class of genes that had been suggested already in the 1980s by others to encode a respiratory enzyme with similarity to the Na+-translocating NADH:quinone oxidoreductase. The rnf genes are widespread in different phylogenetic groups of bacteria and in some archaea. However, detailed knowledge about the function and physiological role of the Rnf complex is scarce. In the last funding period, we characterized the physiological role of Rnf in A. woodii in more detail, addressed the biochemistry and bioenergetics of the activity, and determined properties of the Rnf complex and subunits thereof. Deletion analyses using a bacterium and an archaeon provided evidence that the ferredoxin:NAD oxidoreductase activity is indeed encoded by the rnf genes. We established a reliable enzymatic assay for the complex that allowed unequivocal demonstration of its Na+ dependence and enriched a Na+-Rnf complex from Thermotoga maritima, together with the Na+-ATP synthase. A very important observation was that dicyclohexylcarbodiimide (DCCD) inhibited ferredoxin-dependent NAD reduction and that this inhibition was prevented by preincubation with Na+. In addition, we succeeded in producing proteins homologously in A. woodii which will open the door for a structure-function analysis of the Rnf complex. We will follow up the data obtained during the first funding period and will pursue two major goals. First, the Na+-Rnf and Na+-F1FO ATP synthase preparation described above offers the unique opportunity to test the hypothesis that the Rnf activity is coupled to ion transport and the generation of a membrane potential that then drives ATP synthesis. To this end, Rnf and ATP synthase present in the preparation will be co-reconstituted into liposomes and analyzed biochemically and bioenergetically. Second, the Rnf complex will be characterized with a focus on the subunits and residues involved in ion binding, making use of the competetion of DCCD and Na+. DCCD labeling will be used as a tool to identify the Na+ binding site(s). We have also established recently a procedure to produce proteins in A. woodii. This will allow us to verify the importance of the DCCD-binding site for Na+ transport by mutational analyses and allow to identify other residues of the Na+ binding pocket and even additional Na+-binding pockets. In sum, we will get detailed insights into the function of a novel and so far fairly unexamined respiratory enzyme.