The Dimanganese(II) Site of Bacillus subtilis Class Ib Ribonucleotide Reductase

Class Ib ribonucleotide reductases (RNRs) use a dimanganese-tyrosyl radical cofactor, Mn-2(III)-Y-center dot, in their homodimeric NrdF (beta 2) subunit to initiate reduction of ribonucleotides to deoxyribonucleotides. The structure of the Mn-2(II) form of NrdF is an important component in understanding O-2-mediated formation of the active metal-locofactor, a subject of much interest because a unique flavodoxin, NrdI, is required for cofactor assembly. Bio-chemical studies and sequence alignments suggest that NrdF and NrdI proteins diverge into three phylogenetically distinct groups. The only crystal structure to date of a NrdF with a fully ordered and occupied dimanganese site is that of Escherichia coli Mn-2(II)-NrdF, prototypical of the enzymes from actinobacteria and proteobacteria. Here we report the 1.9 angstrom resolution crystal structure of Bacillus subtilis Mn-2(II)-NrdF, representative of the enzymes from a second group, from Bacillus and Staphylococcus. The structures of the metal clusters in the beta 2 dimer are distinct from those observed in E. coli Mn-2(II)-NrdF. These differences illustrate the key role that solvent molecules and protein residues in the second coordination sphere of the Mn-2(II) cluster play in determining conformations of carboxylate residues at the metal sites and demonstrate that diverse coordination geometries are capable of serving as starting points for Mn-2(II)-Y-center dot cofactor assembly in class Ib RNRs.