Regulation of Electron Transfer Pathways in Cytochromes with Aromatic Amino Acids
Date
4-9-2022
Location
Schewel 2nd Floor Lobby
Description
Natural photosynthetic proteins are based upon chains of reactions that start with sub-nanosecond light energy conversion into the energy of electrical charges and are followed by multi-step electron transfer (ET). Attempts to develop artificial enzymes for photosynthesis have been relatively futile due to the difficulty of generating sufficiently fast primary charge separation even with the smallest proteins. Here we report our results on attempts to accelerate ET by placing aromatic redox-active amino acids along the putative path of ET for the E39C mutant of PpcA, a 3-heme cytochrome. With LC-MS we verified protein purity and successful attachment of Ru(bpy)3 to Cys-39 in E39C, E39C/F41W, E39C/F41Y, and E39C/I38Y mutants. Using analytical size exclusion chromatography, we verified successful protein folding and removal of unreacted photosensitizers. With temperature-dependent circular dichroism spectroscopy, we evaluated the thermal stability of both mutants. Finally, we observed increasing fluorescence quenching under more alkaline conditions in E39C, E39C/F41W, E39C/F41Y, and E39C/I38Y suggesting that Trp-41 and Tyr start to serve as intermediate sites in the ET pathway responsible for the acceleration of ET.
Presentation Type
Poster
Regulation of Electron Transfer Pathways in Cytochromes with Aromatic Amino Acids
Schewel 2nd Floor Lobby
Natural photosynthetic proteins are based upon chains of reactions that start with sub-nanosecond light energy conversion into the energy of electrical charges and are followed by multi-step electron transfer (ET). Attempts to develop artificial enzymes for photosynthesis have been relatively futile due to the difficulty of generating sufficiently fast primary charge separation even with the smallest proteins. Here we report our results on attempts to accelerate ET by placing aromatic redox-active amino acids along the putative path of ET for the E39C mutant of PpcA, a 3-heme cytochrome. With LC-MS we verified protein purity and successful attachment of Ru(bpy)3 to Cys-39 in E39C, E39C/F41W, E39C/F41Y, and E39C/I38Y mutants. Using analytical size exclusion chromatography, we verified successful protein folding and removal of unreacted photosensitizers. With temperature-dependent circular dichroism spectroscopy, we evaluated the thermal stability of both mutants. Finally, we observed increasing fluorescence quenching under more alkaline conditions in E39C, E39C/F41W, E39C/F41Y, and E39C/I38Y suggesting that Trp-41 and Tyr start to serve as intermediate sites in the ET pathway responsible for the acceleration of ET.