Biochemical and electrochemical characterization of redox protein candidates of the iron oxidation in the acidophilic iron oxidizer “Ferrovum” sp. PN-J47-F6

由于电子供体（亚铁/铁）和受体（氧/水）之间极低的氧化还原电位差，将亚铁氧化耦合到氧还原的好氧铁氧化生物在热力学极限下应付生命。由于外部pH也会影响电子供体和受体对的氧化还原电位，从而影响电子传递过程的能量产量，因此可以假设，参与铁氧化的氧化还原蛋白的生化特性也受到栖息地外部pH的影响。然而，对铁氧化剂氧化还原蛋白的生化研究仅限于少数模式生物（Acidithiobacillus ferrooxidans, Ferroplasma ophilum）。然而，基于组学的研究可以预测几种原核生物的电子传递链。Betaproteobacteria中既有嗜酸性铁氧化剂，如Ferrovum，也有嗜中性铁氧化剂。与模式嗜酸菌相比，Ferrovum属的成员是更温和的嗜酸菌。通过基因组分析推断出“Ferrovum”的铁氧化模型，并基于转录组数据进行了扩展。有趣的是，预测铁氧化模型的氧化还原蛋白与属于β -和Zetaproteobacteria的中性铁氧化剂的氧化还原蛋白比与其他嗜酸菌的氧化还原蛋白更相似。因此，“Ferrovum”spp.提供了一个独特的模型来研究铁氧化蛋白的生化特性是否适应了较低的外部ph。因此，本项目旨在研究铁氧化模型候选蛋白的系统发育并表征其生化特性。目的(i)将采用生物信息学方法：计算这些氧化还原蛋白的树形图，然后将其与基于分类标记（即16S rRNA基因序列）的树形图进行比较。目标（二）将在两个层次进行。首先，选择“Ferrovum”sp. PN-J47-F6的可溶性候选蛋白进行异种制备，进行生化和电化学表征，以确定其氧化还原中心并确定其中点电位。蛋白质-蛋白质相互作用的研究计划阐明提出的铁氧化模型的电子传递链。第二阶段涉及“Ferrovum”sp. PN-J47-F6膜组分中野生型蛋白的生化和电化学表征。因此，重组蛋白的生化特性可以作为参考。提出的研究将修正我们目前对微生物铁氧化的认识，提供第一个关于中等嗜酸菌中铁氧化的氧化还原蛋白的生化数据。这些数据可以让我们深入了解这种节能过程的进化是如何受到栖息地外部pH值的影响的。

Aerobic iron oxidizing organisms coupling ferrous iron oxidation to the reduction of oxygen cope with life at the thermodynamic limit due to the extremely low redox potential difference between the electron donor (ferrous/ferric iron) and acceptor couple (oxygen/water). Since external pH also effects the redox potential of the electron donor and acceptor couple and, thereby, the energy yield of the electron transfer process, it has been postulated that the biochemical properties of redox proteins involved in iron oxidation are also effected by the external pH of the habitat. However, biochemical studies on redox proteins of iron oxidizers are limited to few model organisms (Acidithiobacillus ferrooxidans, Ferroplasma acidiphilum). Nevertheless, omics-based research allowed the prediction of electron transfer chains in several prokaryotes. Among Betaproteobacteria there are both acidophilic iron oxidizers, such as “Ferrovum” spp. and neutrophilic iron oxidizers. In comparison to model acidophiles members of the genus “Ferrovum” are more moderate acidophiles. A model of the iron oxidation in “Ferrovum” spp. was inferred by genome analysis and extended based on transcriptome data. Intriguingly, redox proteins of the predicted iron oxidation model share more similarities with those of neutrophilic iron oxidizers belonging to the Beta- and Zetaproteobacteria than with those of other acidophiles.Hence, “Ferrovum” spp. present a unique model to investigate whether biochemical properties of iron oxidation proteins have adapted to the lower external pH. Therefore, the proposed project aims to investigate the phylogeny of the protein candidates of the iron oxidation model and to characterize their biochemical properties. Aim (i) will be approached bioinformatically: Dendrograms of these redox proteins will be calculated and then be compared to dendrograms based on taxonomic markers (i.e. 16S rRNA gene sequence). Aim (ii) will be approached at two levels. First, selected soluble protein candidates of “Ferrovum” sp. PN-J47-F6 will be produced heterologously for biochemical and electrochemical characterization in order to identify their redox centers and to determine their mid-point potential. Protein-protein interaction studies are planned to elucidate the proposed electron transfer chain of the iron oxidation model. The second level involves biochemical and electrochemical characterization of wild type proteins in membrane fractions of “Ferrovum” sp. PN-J47-F6. Therefore, biochemical properties of the recombinant proteins are intended to be used as reference. The proposed study will amend our current knowledge on microbial iron oxidation by providing the first biochemical data on redox proteins involved in iron oxidation in a moderate acidophile. This data may allow valuable insights into how evolution of this energy conserving process has been influenced by external pH of the habitat.