Spectroscopic and computational studies of the de novo designed protein DF2t: correlation to the biferrous active site of ribonucleotide reductase and factors that affect O2 reactivity.

DF2t, a de novo designed protein that mimics the active-site structure of many non-heme biferrous enzymes, has been studied using a combination of circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature variable-field (VTVH) MCD. The active site of DF2t is found to have one five-coordinate iron and one four-coordinate iron, which are weakly antiferromagnetically coupled through a mu-1,3 carboxylate bridge. These results bear a strong resemblance to the spectra of Escherichia coli ribonucleotide reductase (R2), and density functional theory calculations were conducted on the W48F/D84E R2 mutant in order to determine the energetics of formation of a monodentate end-on-bound O2 to one iron in the binuclear site. The mu-1,3 carboxylate bridges found in O2-activating enzymes lack efficient superexchange pathways for the second electron transfer (i.e., the OH/oxo bridge in hemerythrin), and simulations of the binding of O2 in a monodentate end-on manner revealed that the bridging carboxylate ligands do not appear capable of transferring an electron to O2 from the remote Fe. Comparison of the results from previous studies of the mu-1,2 biferric-peroxo structure, which bridges both irons, finds that the end-on superoxide mixed-valent species is considerably higher in energy than the bridging peroxo-diferric species. Thus, one of the differences between O2-activating and O2-binding proteins appears to be the ability of O2 to bridge both Fe centers to generate a peroxo intermediate capable of further reactivity.