Engineering photosensory proteins through the better understanding and control of proton-coupled electron transfer reactions

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A wide range of biological processes make extensive use of electron transfer (ET) reactions, which underlie much of energy transduction and metabolism in the cell. Biological systems are well adapted to direct the movement of electrons among macromolecules while retaining the free energies necessary to drive chemical transformations. ET is often a multistep process in that reactive protein residues act as way stations to harbor electrons or their vacancies during long-range charge migration. Moreover, proton movement is often coupled to ET and the acids and bases in the vicinity of the centers undergoing reduction and oxidation (redox) can exert substantial influence over net reactivity. This project will undertake the study of proton-coupled electron transfer (PCET) in a well-developed model system of protein partners to better understand key parameters of energy transduction and metabolism in biology. Stabilizing charge separation is relevant to energy harvesting strategies, sensing technologies, and the production of stable radical pairs, the latter of which have been implicated in the sensing of Earth’s magnetic field. Better understanding of multi-step PCET in proteins will be exploited in the design of genetically encoded probe molecules and actuators. Control of cofactor redox state with light will enable the application of small fusion proteins as structural probes for cellular studies. The project will couple to an on-going program to advance the success of underserved students and engage them in scientific research. This project aims to study proton-coupled electron transfer (PCET) reactions that involve reactive protein side chains (i.e. hopping sites) to both better understand these essential processes of life and to enable the design of new photosensory proteins to be used as tools in cell and neurobiology. The investigators will apply a photoactive model system composed of zinc-porphyrin-substituted cytochrome c peroxidase and cytochrome c to investigate how management of protonation reactions impact the ability of radical-forming sites to accelerate and steer electron transfer (ET). Kinetic and structural studies will explore the reactivity of tryptophan and tyrosine residues that have been substituted by unnatural derivatives and altered by changes to interacting residues. The properties of the redox partners will be further modulated by controlling their association and their structures and reactivities will be characterized through a variety of spectroscopic and structural approaches. Experiments involving crystals at high-pressure will potentially reveal the subtle dependence of PCET on detailed bonding networks. Through both rational and selection methods the investigators will optimize photoreduction of flavin-binding Light- Oxygen- Voltage (LOV) photoreceptors to generate new genetically encoded probes for in vivo electron-spin resonance spectroscopy applications. Control of flavin PCET will also enable the design of new cryptochrome proteins that sense in altered regions of the spectrum and drive interactions that can be adopted for optogenetics. In exploring these systems, the investigators will take a comprehensive mechanistic approach that includes enzymology, spectroscopy, structural biology and computation.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.