Electron Transfer to Fe(III) in the Geobacteraceae

本研究的目的是阐明异化的铁（III）还原微生物硫还原Geobacter sulphreducens中电子向铁（III）的传递机制。G. sulphreducens是变形菌门三角洲亚类（Geobacteraceae）微生物家族的代表，它具有保存能量的能力，以支持有机化合物的氧化和铁（III）的还原。之所以选择G.硫还原菌进行这些研究，是因为它易于大众培养，并应适用于未来的遗传研究。最初的研究将集中在nadh依赖的Fe（III）还原酶复合物上，该复合物位于G.硫还原酶的膜部分。Fe（III）还原酶复合物将被纯化到足够的量，以表征最佳活性、底物亲和力、亚基组成、金属含量和氧化还原电位的条件。复合体中亚基的基因将被克隆并测序。将进行分解和重构研究，以确定纯化为nadh依赖性铁（III）还原酶复合体的所有五种蛋白质是否实际上是铁（III）还原所必需的。特别强调的是确定哪一种蛋白质负责将电子转移到Fe（III）。铁（III）还原酶将使用细胞免疫学技术定位在细胞内，以确定它是否位于外膜，这将被认为是一种将电子传递到细胞外铁（III）的酶。将进行研究，其中铁（III）还原酶复合物的基本成分将被纳入人工膜中，以确定当铁（III）还原酶成分与膜结合时，铁（III）是否以生理相关的速率还原。一旦从NADH到Fe（III）的电子传递机制被阐明，参与h2依赖的Fe（III）还原的电子传递组分将被研究。这将包括确定G.硫还原中哪一种氢化酶能够为Fe（III）还原提供电子，在H2氧化中起作用的Fe（III）还原酶是否与NADH氧化相同，以及确定Fe（III）还原所必需的其他成分。这些研究有望对微生物将有机物氧化耦合到铁（III）还原的机制提供基本的理解。最近人们认识到，微生物可以通过氧化有机化合物并减少Fe（III）来保存能量，在全球碳循环和水生沉积物和地下水有机污染的修复中发挥重要的环境作用。铁（III）还原微生物可以在其代谢中代替有毒金属铁（III），因此也可以帮助修复金属污染的环境。此外，地质证据表明，Fe（III）还原是古代地球上第一个将有机物完全氧化回二氧化碳的全球性重要过程。因此，阐明微生物将电子转移到Fe（III）的机制将为这一重要的微生物代谢形式提供重要的基础见解，更好地理解微生物呼吸的进化，并有可能推进这一代谢途径在环境恢复中的应用。

9727840 Lovley The objective of this study is to elucidate the mechanisms for electron transport to Fe(III) in the dissimilatory Fe(III)-reducing microorganism, Geobacter sulfurreducens. G. sulfurreducens is representative of a family of microorganisms in the delta subclass of the Proteobacteria, the Geobacteraceae, which have the capacity to conserve energy to support growth from the oxidation of organic compounds coupled to the reduction of Fe(III). G. sulfurreducens has been chosen for these studies because it is easy to mass culture and should be amenable to future genetic studies. Initial studies will focus on the NADH-dependent Fe(III) reductase complex that is localized in the membrane fraction of G. sulfurreducens. The Fe(III) reductase complex will be purified in sufficient quantities to characterize conditions for optimal activity, substrate affinities, subunit composition, metal content, and redox potentials. The genes for the subunits in the complex will be cloned and sequenced. Disaggregation and reconstitution studies will be conducted to determine if all of the five proteins that purify as the NADH-dependent Fe(III) reductase complex are actually necessary for Fe(III) reduction. Special emphasis will be placed on identifying which of the proteins is responsible for transferring electrons to Fe(III). The Fe(III) reductase will be localized in the cell using cytoimmunological techniques in order to determine if it is in the outer membrane as would be expected for an enzyme that is considered to pass electrons to extracellular Fe(III). Studies will be conducted in which the essential components of the Fe(III) reductase complex will be incorporated into artificial membranes to determine if Fe(III) is reduced at physiologically relevant rates when the Fe(III) reductase components are membrane-bound. Once the mechanisms for electron transport from NADH to Fe(III) are elucidated, the electron transport components involved in H2-dependent Fe(III) reduction will be studied. This will involve determining which of the hydrogenases in G. sulfurreducens is capable of donating electrons for Fe(III) reduction, whether the Fe(III) reductase that functions in H2 oxidation is the same as for NADH oxidation, and determining what other components are necessary for Fe(III) reduction. These studies are expected to provide a fundamental understanding of the mechanisms by which microorganisms couple the oxidation of organic matter to the reduction of Fe(III). It has recently been recognized that microorganisms that can conserve energy by oxidizing organic compounds with the reduction of Fe(III) play an important environmental role in the global carbon cycle and in the remediation of organic pollution of aquatic sediments and groundwater. Fe(III)-reducing microogranisms can substitute toxic metals for Fe(III) in their metabolism and thus can also aid in the remediation of metal-contaminated environments. Furthermore, geological evidence suggests that Fe(III) reduction was the first globally significant process for completely oxidizing organic matter back to carbon dioxide on ancient earth. Thus, elucidating the mechanisms by which microorganisms transfer electrons to Fe(III) will provide important basic insights into this important form of microbial metabolism, provide a better understanding of the evolution of microbial respiration, and is likely to advance the application of this metabolic pathway to environmental restoration.