Divergent assembly mechanisms of the manganese/iron cofactors in R2lox and R2c proteins.

A manganese/iron cofactor which performs multi-electron oxidative chemistry is found in two classes of ferritin-like proteins, the small subunit (R2) of class Ic ribonucleotide reductase (R2c) and the R2-like ligand-binding oxidase (R2lox). It is unclear how a heterodimeric Mn/Fe metallocofactor is assembled in these two related proteins as opposed to a homodimeric Fe/Fe cofactor, especially considering the structural similarity and proximity of the two metal-binding sites in both protein scaffolds and the similar first coordination sphere ligand preferences of Mn and Fe. Using EPR and Mössbauer spectroscopies as well as X-ray anomalous dispersion, we examined metal loading and cofactor activation of both proteins in vitro (in solution). We find divergent cofactor assembly mechanisms for the two systems. In both cases, excess Mn promotes heterobimetallic cofactor assembly. In the absence of Fe, R2c cooperatively binds Mn at both metal sites, whereas R2lox does not readily bind Mn at either site. Heterometallic cofactor assembly is favored at substoichiometric Fe concentrations in R2lox. Fe and Mn likely bind to the protein in a stepwise fashion, with Fe binding to site 2 initiating cofactor assembly. In R2c, however, heterometallic assembly is presumably achieved by the displacement of Mn by Fe at site 2. The divergent metal loading mechanisms are correlated with the putative in vivo functions of R2c and R2lox, and most likely with the intracellular Mn/Fe concentrations in the host organisms from which they were isolated.