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The Geobacter bacteria can oxidize petroleum compounds and have already shown a significant potential for harvesting the energy from organic compounds into electricity in microbial fuel cells and for environmental cleanup of petroleum spills. However, little is known about the chemical mechanisms that govern redox activity of these bacteria. Recently discovered heme sensors GSU0582 and GSU0935 from Geobacter sulfurreducens switch their axial ligand H₂O to Met60 upon reduction of the heme iron. This change in the metal coordination environment is expected to affect the redox reactivity of the heme and could also require changes in the protein conformation. However, the mechanistic details of such redox transformations in Geobacter cytochromes as well as other ligand-switching redox proteins are largely unknown. We aim to probe electron-transfer (ET) reactivity, ligand-substitution dynamics and other conformational processes in GSU0582 and GSU0935 in order to determine the nature and origin of conformational control in these bioinorganic systems. These studies will not only explore important problems in coordination chemistry and ET mechanisms but could also provide valuable insights for engineering microbial fuel cells and design of molecular systems with switchable ET properties for solar-energy harvesting.

We have designed an experimental system to examine reduction and accompanying conformational changes associated with the H₂O-Met ligand switch in GSU0582 and GSU0935. The reduction is driven by a photoexcitation a covalently attached Ru(bipy)₂(phenanthroline)²⁺ complex, which creates strong reducing agent, *Ru²⁺. The *Ru²⁺ species rapidly reduces the heme and the resulting conformational changes can be followed by kinetic methods. The redox-linked ligand switching at the heme is also detectable in bimolecular gated redox reactions, both thermal and photoinduced.

In this first year of funding, our research was focused on preparation of GSU0935 mutants and their Ru-labeled derivatives, as well as spectroscopic studies of redox reactions and folding of the wild-type GSU0935. We have synthesized the thiol-specific Ru-labeling reagent and expressed and purified three different cysteine mutants of GSU0935 for coupling with this tag. One of the mutants, G58C, was labeled with Ru-bipyridyl tag and preliminary kinetic results were obtained. We have also prepared several mutants of cytochrome c to serve as models of differently ligated hemes. The redox-induced changes in the wild-type GSU0935 have been examined with photogenerated ³Zncyt and by direct electrochemistry. The laser-flash photolysis studies have detected a redox-linked conformational step. Electrochemistry studies, done in collaboration with Sean Elliott’s group, have confirmed that redox reactions of GSU0935 are gated. Unfolding reactions of GSU0935 were studied with the protein in reduced and oxidized state. Relatively slow unfolding of the ferric GSU0935 suggests a distinct step that gates protein unfolding. We are currently exploring if the same structural transition plays a role in the redox mechanism of the protein. Our electron-transfer work on Geobacter cytochromes offers useful methodology for studies of other proteins where redox-linked conformational changes regulate biochemical pathways.

This project has impacted the careers of the involved researchers in the following ways. (1) The research has served as a training ground in molecular biology, spectroscopy, and inorganic chemistry for several undergraduate researchers and a first-year graduate student. (2) The undergraduate summer research on this project was covered in Nucleus, a newsletter of the Northeast Section of ACS. (3) The project initiated collaboration with Elliott’s group at Boston University; exchange of research findings has enhanced scientific experience of students in both groups. (4) The PRF funding has allowed us to hire a postdoc who will work on this project during the next year of funding. (5) The PI has presented results of this research at the 2010 Metals in Biology GRC.