PROJECT SUMMARY/ABSTRACT Class Ia ribonucleotide reductases (RNRs) are anti-cancer and antibiotic drug targets due to their role in the biosynthesis of deoxyribonucleotides. They convert ribonucleotides to deoxyribonucleotides using radical- based chemistry with reducing equivalents coming from a pair of cysteine residues in the active site that form a disulfide upon enzyme turnover. Recent structural work elucidating the active state structure of the class Ia RNR of Escherichia coli has revealed for the first time, the intact 32-Å-long pathway for radical transfer between subunits that is responsible for the generation of a catalytically essential radical species in the enzyme active site. In this proposed research, I will investigate the role of water channels in the proton transfer that occurs concomitant with electron transfer. I will also investigate the role of a tryptophan residue (W48) in a radical transfer pathway that otherwise involves tyrosine residues. Furthermore, I will probe the structural rearrangements that occur in order to facilitate re-reduction of the active site disulfide. This re-reduction pathway is known to be dependent on the C-terminal tail of the α subunit of RNR, which shuttles reducing equivalents from thioredoxin to the active site, but no structural snapshots of this process have ever been captured. Here, I propose to use site-specific mutagenesis to investigate the involvement of water channels in proton transfer by assessing the catalytic competency of RNR variants in which the water networks have been disrupted. I intend to assess the role of W48 in the PCET pathway through the site-specific incorporation of unnatural W analogues which will give us a spectroscopic handle with which to directly monitor radical transfer to and from W48. Finally, I propose to capture structural snapshots of the re-reduction steps of RNR, including a structure of RNR with thioredoxin using cryogenic electron microscopy. These studies will enable us to answer several of the major outstanding questions of this prototypical RNR. This work will also enable me, a physical inorganic and materials chemist by training, to gain expertise in biochemical and structural biology methods which will inform new research directions that will benefit from a synthesis of these two skill sets. As one of the world’s foremost research institutions, MIT is an ideal and highly collaborative environment in which to pursue the studies described in this proposal. Furthermore, as an internationally recognized leader in the field of structural biology, Prof. Drennan’s scientific mentorship will enable me to excel in my undertaking of this research.