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.
