Electron Transfer to Fe(III) in the Geobacteraceae

地杆菌科中电子转移至 Fe(III)

• 批准号: 9727840
• 负责人: Derek Lovley
• 金额: $ 29.98万
• 依托单位: University of Massachusetts Amherst
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-04-15 至 2002-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9727840&HistoricalAwards=false
• 关键词: Electron; Transfer; Fe; III; Geobacteraceae

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.

9727840 Lovley本研究的目的是阐明异化还原Fe(III)微生物--硫还原地杆菌中电子向Fe(III)传递的机制。硫磺还原菌是变形杆菌、地杆菌科三角洲亚类中一类微生物的代表，它们具有保存能量以支持有机化合物氧化与Fe(III)还原相结合的生长的能力。硫磺还原菌被选择用于这些研究，因为它易于大规模培养，并应服从于未来的遗传研究。最初的研究将集中在硫还原革兰氏菌膜组分中依赖NADH的Fe(III)还原酶复合体上。Fe(III)还原酶复合体将得到足够数量的纯化，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。该复合体中各亚单位的基因将被克隆和测序。将进行解聚和重组研究，以确定被纯化为依赖NADH的Fe(III)还原酶复合体的所有五种蛋白质是否实际上都是Fe(III)还原所必需的。将特别强调确定哪种蛋白质负责将电子转移到Fe(III)。Fe(III)还原酶将使用细胞免疫学技术定位在细胞内，以确定它是否像被认为将电子传递到细胞外Fe(III)的酶那样位于外膜。将进行将Fe(III)还原酶复合体的基本成分结合到人工膜中的研究，以确定当Fe(III)还原酶组分被膜结合时，Fe(III)是否以生理上相关的速率被还原。一旦阐明了电子从NADH到Fe(III)的传递机制，就将研究依赖于H2的Fe(III)还原所涉及的电子传递组分。这将涉及确定硫还原菌中的哪些氢酶能够为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)还原需要哪些其他成分。这些研究有望为微生物将有机物的氧化与Fe(III)的还原相结合的机制提供一个基本的理解。最近人们认识到，微生物可以通过氧化有机化合物和还原Fe(III)来节约能源，在全球碳循环和修复水中沉积物和地下水的有机污染方面发挥着重要的环境作用。还原Fe(III)的微生物可以在代谢过程中替代有毒金属Fe(III)，从而有助于修复被金属污染的环境。此外，地质证据表明，在古代地球上，Fe(III)还原是第一个在全球范围内将有机物完全氧化回二氧化碳的重要过程。因此，阐明微生物将电子传递给Fe(III)的机制将为了解微生物代谢的这一重要形式提供重要的基础认识，有助于更好地了解微生物呼吸的进化，并有可能推动这一代谢途径在环境修复中的应用。