Understanding multistep electron transfer reactions for the design of photsensory proteins

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In biological systems, many metabolic processes involve electron transfer (ET) reactions, which function to direct the movement of electrons among macromolecules without dissipating the high free energy needed to drive chemical transformations. ET is often multi-step in that reactive protein residues act as waystations to harbor electrons or their vacancies ('holes') during long-range charge migration. Understanding this so-called electron, or hole, 'hopping' is important for adapting key properties of proteins for the purpose of energy conversion. This project will apply spectroscopic and structural techniques to study the role of multi-step ET in model systems and in native biological light sensors to test mechanisms and design new functionality. These studies also will have important prospects for optogenetics, the development and use of engineered photosensitive proteins for controlling cellular processes, including circadian rhythms, with light. The project will involve graduate and undergraduate students in cutting edge research in fields including enzymology, protein biochemistry, structural biology, spectroscopy, chemical kinetics, and protein engineering. The collaborative and multidisciplinary nature of the project will expose students to research that bridges disciplines and scientific cultures. The project will also couple to an on-going program developed by the PI to engage students from under-represented groups in scientific research. This project aims to understand multi-step ET reactions that involve reactive protein side chains (i.e., hopping) in photoactive proteins. Kinetic and structural studies of a photosensitive model system composed of redox partners cytochrome c peroxidase and cytochrome c will explore the reactivity of a Tryptophan/Tyrosine hopping center by systematic perturbation of the radical environment and incorporation of non-natural amino acids at the hopping site. Coupled work on hole-hopping in blue-light sensing cryptochromes and Light- Oxygen- Voltage (LOV) photoreceptors will probe the biophysical attributes that allow reactive protein side chains to mediate rapid and stable charge separation in these flavin-containing photosensors. For a new class of LOV-like protein, multi-step ET involving non-aromatic residues will be investigated. Redesign of these systems will test hypotheses, confirm mechanisms and develop tools for applications of optogenetics, the field of engineering cellular systems to be regulated by light.