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)和受体对(氧/水)之间极低的氧化还原电势差，将亚铁氧化与氧还原耦合在一起的好氧铁氧化生物在热力学极限下应对生命。由于外界pH也影响电子供体和受体对的氧化还原电势，从而影响电子传递过程的能量产率，因此推测参与铁氧化的氧化还原蛋白质的生化性质也受到生境外部pH的影响。然而，对氧化铁菌氧化还原蛋白的生化研究仅限于少数模式生物(氧化亚铁硫杆菌、嗜酸铁质)。然而，基于组学的研究允许预测几种原核生物中的电子转移链。在贝塔变形杆菌中，既有嗜酸性铁氧化剂，如“铁腐菌”。以及中性铁氧化剂。与模型嗜酸菌相比，“Ferrovum”属的成员是更中等的嗜酸菌。铁细菌铁氧化作用的一个模型。是通过基因组分析推断的，并基于转录组数据进行了扩展。有趣的是，所预测的铁氧化模型的氧化还原蛋白与属于β-和Zeta蛋白细菌的中性铁氧化菌的氧化还原蛋白比与其他嗜酸性细菌的氧化还原蛋白更相似。因此，“Ferrovum”spp.提出了一个独特的模型来研究铁氧化蛋白的生化特性是否已经适应了较低的外界pH。因此，拟议的项目旨在调查铁氧化模型候选蛋白质的系统发育，并表征它们的生化特性。目的(I)将从生物信息学的角度进行探讨：计算这些氧化还原蛋白的树状图，然后与基于分类标记(即16S rRNA基因序列)的树状图进行比较。目标(二)将在两个层面上进行。首先，筛选出“Ferrovum”sp.的候选可溶性蛋白。将异源生产PN-J47-F6用于生化和电化学表征，以确定其氧化还原中心和确定其中点电位。计划进行蛋白质-蛋白质相互作用研究，以阐明所提出的铁氧化模型的电子转移链。第二个水平涉及“Ferrovum”sp.膜组分中野生型蛋白的生化和电化学性质。PN-J47-F6。因此，重组蛋白的生化性质可供参考。这项拟议的研究将修正我们目前关于微生物铁氧化的知识，提供有关中等嗜酸菌中参与铁氧化的氧化还原蛋白的第一个生化数据。这一数据可能有助于我们深入了解栖息地的外部酸碱度是如何影响这种能量守恒过程的演化的。

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.