B02 - Reversible Long-range PCET in Ribonucleotide Reductases

Proton-coupled electron transfer is emerging as a fundamental mechanism in biological transformations. A paradigmatic model for biological proton-coupled electron transfer (PCET) is constituted by class I ribonucleotide reductases (RNRs), the enzymes which catalyse the formation of the DNA building blocks in all living cells. In these enzymes, a tyrosyl radical in the protein β-subunit activates the catalytic site in the corresponding α-subunit over an unprecedented long distance of up to 35 Å, through the formation of three transient tyrosyl radical intermediates. Progress in biochemical methods (site-selective insertion of unnatural amino acids, UAA) combined with EPR and transient optical spectroscopy, structure analysis by protein crystallography and single-particle cryo-EM has started to unravel the molecular details of this highly sophisticated mechanism, the disruption of which would inevitably lead to cell death. The goal of this project is to take advantage of previously devised methods and to develop novel techniques to acquire fundamental information on physicochemical and structural parameters which underlie biological PCET in RNR. We will employ tailored experiments to detect distances and trace the transferred protons in PCET steps as a function of critical physicochemical parameters as e.g. buffer pH and pKa values of involved amino acids. The studies will be enabled by a combined EPR spectroscopic and biochemical approach, and will be supported by additional biophysical studies such as high-resolution protein crystallography and single-particle cryo- EM. The results will serve as a basis for quantitative theoretical models and predictions as well as a starting point to tune and control PCET mechanisms in biological molecules. On the short term, we will focus on the most intriguing PCET step across the α/β-subunit interface of the bacterial RNR. Advanced hyperfine spectroscopy for the detection of distant magnetic nuclei (for instance 17O, 15N and 13C) in the second ligation sphere will be explored. The EPR spectroscopic analysis will be accompanied by steady-state and transient kinetics as well as structure determination of RNR in different functional states at very high resolution.