Mechanistic studies of the cofactor assembly in class Ib ribonucleotide reductases and protein affinity for MnII and FeII.

Ribonucleotide reductase (RNR) is an essential enzyme found in all organisms. The function of RNR is to catalyze the conversion of nucleotides to deoxynucleotides. RNRs rely on metallo-cofactors to oxidize a conserved cysteine in the active site of the enzyme into a thiyl radical, which then initiates nucleotide reduction. The proteins required for MnIII2-Y• cluster formation in class Ib RNR are NrdF (β-subunit) and NrdI (flavodoxin). An oxidant is channeled from the FMN cofactor in NrdI to the dimanganese center in NrdF, where it oxidizes the dimanganese center and a tyrosyl radical (Y•) is formed. Both S. sanguinis and E. coli MnII2-NrdF structures have a constriction in the channel immediately above the metal site. In the E. coli, the constriction is formed by the side chain of S159, whereas in the S. sanguinis system it involves T158. This serine to threonine substitution was investigated using S. sanguinis and S. pnemoniae class Ib RNRs but it is also present in other pathogenic streptococci. Using stopped-flow kinetics we investigate the role of this substitution on the mechanism of MnIII2-Y• cluster formation. In addition to different kinetics observed in the studied streptococci, we found that affinity constants of NrdF for MnII and FeII are about 1 µM and the previously reported preference for MnII could not be explained by affinity only.