Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2012.Cataloged from PDF version of thesis.Includes bibliographical references.Ribonucleotide reductases (RNRs) catalyze the reduction of nucleotides to deoxynucleotides in all organisms. The class Ia and lb RNRs comprise two subunits: a2 contains the site of nucleotide reduction, and p2 contains an essential stable tyrosyl radical (Y·), generated by oxidation of a dinuclear metal cluster. The diferric-Y (Fe" 2-Y·) cofactor of the class Ia RNRs self-assembles by reaction of Fe"2-NrdB with 02 and a reducing equivalent. Whether the class Ib RNRs utilize a diiron or dimanganese cofactor in vivo has been controversial. To determine the physiological metallocofactor of the Escherichia coli class lb RNR, we recombinantly express and purify a2 (NrdE) and p2 (NrdF) and show that NrdF self-assembles an active Fe 12- Y· cofactor using Fe" and 02. We also present the first purification of NrdI, a protein of unknown function conserved in class lb RNR systems. We show that NrdI is a flavodoxin-like protein with unusual redox properties. Although Mnr 2-NrdF does not react with 02, in the presence of reduced NrdI (Nrdlhq) and 02, it assembles an active dimanganese(III)-Y· (Mn 12- Y·) cofactor. Biochemical evidence indicates that Nrdlhq binds tightly to NrdF and reacts with 02 to provide an oxidant that channels to the metal site in NrdF to assemble the Mn"12-Ycofactor, a model supported by crystal structures of a Mn"2-NrdF*NrdI complex. NrdF purified from its endogenous levels in an iron-limited E. coli strain contains the Mn" 2 -Y· cofactor, establishing its physiological relevance. Rapid kinetics studies of Mn"'12 -Y· cofactor assembly in Bacillus subtilis NrdF support a mechanism in which NrdIhg rapidly reduces 02 to 02- and the 02'- channels to and reacts with Mn"2-NrdF to form a Mn" Mnv intermediate, which oxidizes tyrosine to Y·. Finally, we also demonstrate that E. coli NrdF, when incubated anaerobically with Mn" and Fe" and then exposed to H202 , forms an active Y·-containing metallocofactor that we suggest is Fe"Mn'l-Y·. These results raise the issues of how a single active site can generate a stable, active Ye using three different metal cofactors and oxidants in vitro, and therefore how metallation of NrdF with manganese is controlled in vivo.by Joseph Alfred Cotruvo, Jr..Ph.D