Mechanistic studies of the cofactor assembly in class Ib ribonucleotide reductases and protein affinity for Mn^II and Fe^II

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 metallocofactors 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 Mn^III₂–Y^• cluster formation in class Ib RNRs 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 Streptococcus sanguinis and Escherichia coli Mn^II₂–NrdF structures have a constriction in the channel immediately above the metal site. In 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 Streptococcus pneumoniae class Ib RNRs but it is also present in other pathogenic streptococci. Using stopped-flow kinetics, we investigate the role of this substitution in the mechanism of Mn^III₂–Y^• cluster formation. In addition to different kinetics observed in the studied streptococci, we found that affinity constants of NrdF for Mn^II and Fe^II are about 1 μM and the previously reported preference for Mn^II could not be explained by affinity only.