Reports: DNI349953-DNI3: Ligand Substitution and Conformational Control of a Metal-Site Redox Function

Ekaterina Pletneva, PhD , Dartmouth College

The Geobacter bacteria can oxidize petroleum compounds and have already shown a 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. 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 are probing electron-transfer (ET) reactivity, ligand-substitution dynamics and other conformational processes in GSU0582 and GSU0935 in order to determine the nature 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 examine photoinduced and thermal redox reactions and accompanying conformational changes associated with the H2O-Met ligand switch in GSU0582 and GSU0935. A covalently attached Ru(bipy)2(phenanthroline)2+ complex and zinc-substituted porphyrin in horse heart cytochrome c, Zncyt, serve to generate powerful reductants in the respective photoinduced unimolecular and bimolecular redox reactions. The ligand switching at the heme gates the redox reaction and is detectable both in kinetic and electrochemistry measurements. Folding studies suggest the role of ligand substitution during protein refolding further illustrating the flexibility of the heme environment in these unusual cytochromes.

In this year of funding, our research was focused on preparation of GSU0935 mutants, their fluorescent and Ru-labeled derivatives, as well as spectroscopic studies of redox reactions and folding of the wild-type GSU0935. We have expressed and purified several mutants of GSU0935 for site-specific placement of photoactive probes and for eliminating ligand substitution (in the Met60Ala/Met61Val mutant). The redox reactions in the wild-type GSU0935 have been examined with photogenerated 3Zncyt. Absorption and resonance Raman studies excluded the presence of Met-coordinated species in ferric GSU0935. Refolding of the high-spin ferric GSU0935 proceeds through a low-spin intermediate suggesting the role of ligand substitution in this process as well. We are currently working to identify the nature of the intermediate ligand and the sequence of refolding steps with fluorescence approaches. The solvent-exposed hemes confer high peroxidase activity to these folded Geobacter proteins, which may yield interesting applications for catalysis.

Difficulties in large-scale expression of some of the GSU0935 mutants prompted us to search for better-behaved models of ligand-switching hemes. We have prepared several mutants of iso-1 yeast cytochrome c with different ligands at the heme and a mutant that undergoes His-to-Met ligand switching upon reduction. Flash-photolysis and computational studies of redox reactivity of these mutants are underway. We are also investigating conformational properties and redox-dependent folding stability of the switchable F82H cytochrome c mutant.

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 an undergraduate researcher, two graduate students, and a postdoc. (2) The project involved collaboration with Elliott’s group at Boston University; exchange of research findings has enhanced scientific experience of students in both groups. (3) The PRF funding has allowed us to support a postdoc who will continue work on this project  and prepare results for publication during the next year of funding. (4) The project results, laser spectroscopy setup, and student training served as a foundation for new group projects now supported by NIH and NSF. (5) The PI has presented results of this research during her seminar trips and informally during discussions with colleagues at the 2011 Metals in Biology Gordon Research Conference and 241st ACS meeting in Anaheim.

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