Investigation of Fe(III) Reduction in Geobacter Sulfurreducens Characterization of Outer Surface Associated Electron Transfer Components
Date of Award
Open Access Dissertation
Doctor of Philosophy (PhD)
Derek R. Lovely
Michael J. Maroney
Outer membrane cytochromes OmcB and OmcS of Geobacter sulfurreducens are two important components of the respiratory chain for extracellular Fe(III) reduction. OmcS is a loosely bound cell surface protein involved in the reduction of insoluble Fe(III). OmcB is an outer membrane protein and required for insoluble and soluble Fe(III) reduction. The objective of this study was to understand better the mechanism of dissimilatory Fe(III) reduction, focusing on the cell surface proteins by further localization, identification of protein-protein interactions, and biochemical characterization of OmcB and OmcS. OmcB was found to be surface-exposed but embedded in the outer membrane because mild protease treatment of cells resulted in partial degradation of OmcB. Removal of surface-exposed proteins inhibited Fe(III) reduction, which is at least partially due to the degradation of OmcB. Co-immunoprecipitation studies with outer surface proteins using an antibody against OmcS revealed that OmcS interacts with several proteins, of which some are implicated in Fe(III) reduction, such as PilA, OmpJ, and OmpB, and in electricity production, such as OmcZ. Other OmcS-associated proteins, which have not been studied, include a cytochrome (GSU2887), a hypothetical and a conserved hypothetical protein, and a putative protease with a PDZ domain. The results suggest that co-immunoprecipitation with other antibodies would help to identify more elements of electron transport pathways related to extracellular Fe(III) reduction. OmcB was purified via preparative sodium dodecylsulfate polyacrylamide gel electrophoresis (SDSPAGE) and anion-exchange chromatography. The molecular mass was determined as 82 kDa, and 11.5 hemes per molecule were found. OmcB was able to transfer electrons to either soluble or insoluble Fe(III). OmcS was purified by detergent extraction. The molecular mass was 47 kDa and it contains 6 heme groups. UV-visible, EPR, and NMR spectroscopies determined that all hemes are bis-histidyl hexacoordinated and low-spin in both oxidized and reduced forms. OmcS has a –212 mV midpoint redox potential, and donates electrons to soluble and insoluble metals and quinones. Transient state kinetics showed that OmcS reduces anthroquinone-2, 6-disulfonate 10 times faster than it reduces Fe(III) citrate. This study revealed valuable further details about the mechanism of Fe(III) reduction by G. sulfurreducens by identifying the localization, protein-protein interactions and biochemical characteristics of the components of extracellular electron transport.
Qian, Xenlei, "Investigation of Fe(III) Reduction in Geobacter Sulfurreducens Characterization of Outer Surface Associated Electron Transfer Components" (2009). Open Access Dissertations. 116.