Crystal structure of the ribonucleotide reductase R2 mutant that accumulates a mu-1,2-peroxodiiron(III) intermediate during oxygen activation

The R2 subunit of Escherichia coli (aerobic) ribonucleotide reductase activ ates molecular oxygen at its diiron center to produce a functionally essent ial stable tyrosyl radical from residue Y122. It was previously shown that the D84E site-directed mutant of R2(R2-D84E) accumulates a mu-1,2-peroxodii ron(III) intermediate on the pathway to tyrosyl radical formation. This int ermediate does not accumulate in the reaction of wildtype (wt) R2, but an a nalogous complex does accumulate during oxygen activation by the structural ly similar diiron protein, methane monooxygenase hydroxylase (MMOH). Herein we describe the crystallographically determined three-dimensional structur es of the reduced, diiron(II) reactant and oxidized, diiron(III) product fo rms of R2-D84E. The reduced R2-D84E structure differs from that of reduced wt R2 in the conformations of three carboxylate ligands, E84, E204, and E23 8. The adjustments in these ligands render the coordination sphere of the d iiron(II) center very similar to that in reduced MMOH. In addition, a water molecule not observed in reduced wt R2 is coordinated to Fe2 in reduced R2 -D84E. The oxidized R2-D84E structure is similar to that of oxidized wt R2 except in the coordination mode of E84. In R2-D84E, E84 coordinates to Fel in a monodentate, terminal mode and is hydrogen bonded to a water molecule also coordinated to Fel. In wt R2, D84 is a bidentate, chelating ligand. In both R2-D84E structures, Y122 is shifted away from Fel such that a hydroge n bonding interaction with E84 is not possible. The observed structural adj ustments suggest possible rationales for the stability of the mu-1,2-peroxo diiron(III) complex in R2-D84E. In addition, the structures expand the expe rimental foundation for computational investigations aimed at defining the detailed mechanistic pathways for O-2 activation at diiron(II) centers.