Project Summary/Abstract Controlling the movement of electrons and protons is critical for a wide range of biological processes, including cellular respiration, DNA biosynthesis, and photoreception used for optogenetics. Many of these processes are driven by the formation of tyrosine or tryptophan radical species via proton-coupled electron transfer (PCET). An elementary PCET reaction involves the transfer of one electron and one proton, but more complex PCET processes involve the transfer of multiple electrons and protons. The Hammes-Schiffer group has developed a general PCET theory enabling the calculation of rate constants and has applied this theory to biomimetic model systems and to elementary PCET in an enzyme. Simulating more complex biological PCET processes is challenging because of the significance of hydrogen tunneling and conformational motions, as well as key contributions from multiple time and length scales. A major goal of this proposal is to develop a multiscale modeling approach that describes the individual PCET steps, including the electronic and nuclear quantum effects, as well as the key conformational changes coupled to them. Quantum chemistry and molecular dynamics methods will be used to compute the input quantities to the PCET theory. The calculated rate constants for individual PCET reactions and protein conformational changes will serve as input into microkinetic models to enable the complete description of complex multi-electron, multi-proton biological processes. This multiscale modeling approach will be closely connected to experimental data, relying on atomic-level structures and thermodynamic and kinetic measurements. Initially this approach will be applied to PCET in the well-defined, controlled protein environment of the α3X proteins, which consist of three alpha helices with a single interior tyrosine or tryptophan that can be oxidized electrochemically. This approach will be expanded to explore multi- electron, multi-proton reactions in the more complex protein environment of ribonucleotide reductase (RNR). This enzyme catalyzes the conversion of nucleotides to deoxynucleotides, thereby maintaining the nucleotide pool balance required for effective DNA synthesis, replication, and repair. In addition to its biochemical importance, RNR serves as a prototype for multi-step biological PCET. The long-range radical translocation over ~35 Å in RNR is proposed to occur via a series of PCET steps involving tyrosine and tryptophan residues, as well as significant conformational changes. A recently solved cryo-EM structure of the active complex resolves the entire PCET pathway and provides an opportunity for theoretical studies. This work will elucidate the impact of the protein electrostatic environment, solvent accessibility, and conformational motions on PCET. It will also provide insights into how individual PCET steps are coupled to each other and to protein conformational motions and what determines the order of the steps and the overall rate. Discovering the factors that impact biological PCET is vital for understanding and controlling a wide range of essential biochemical processes. These fundamental insights may also have broader implications for protein design and optogenetics.

Project Narrative This MIRA project will use theoretical methods, closely coupled with experimental data, to elucidate the basic factors that control the movement of electrons and protons in biological systems, providing fundamental insights into a wide range of biochemical processes essential to living systems. In particular, this project will enhance understanding of the radical translocation mechanism of ribonucleotide reductase, which plays a critical role in DNA synthesis, replication, and repair. The general insights about controlling electrons and protons provided by this work may also have broader implications for protein engineering, drug design, and optogenetics.