Composition and Structure of the Inorganic Core of Relaxed Intermediate X (Y122F) of Escherichia coli Ribonucleotide Reductase

Activation of the diferrous center of the β2 (R2) subunit of the class 1a Escherichia coli ribonucleotide reductases by reaction with O₂ followed by one-electron reduction yields a spin-coupled, paramagnetic Fe(III)/Fe(IV) intermediate, denoted X, whose identity has been sought by multiple investigators for over a quarter of a century. To determine the composition and structure of X, the present study has applied ⁵⁷Fe, ^14,15N, ¹⁷O, and ¹H electron nuclear double resonance (ENDOR) measurements combined with quantitative measurements of ¹⁷O and ¹H electron paramagnetic resonance line-broadening studies to wild-type X, which is very short-lived, and to X prepared with the Y122F mutant, which has a lifetime of many seconds. Previous studies have established that over several seconds the as-formed X(Y122F) relaxes to an equilibrium structure. The present study focuses on the relaxed structure. It establishes that the inorganic core of relaxed X has the composition [(OH^-)Fe^III-O-Fe^IV]: there is no second inorganic oxygenic bridge, neither oxo nor hydroxo. Geometric analysis of the ¹⁴N ENDOR data, together with recent extended X-ray absorption fine structure measurements of the Fe-Fe distance (Dassama, L. M.; et al. J. Am. Chem. Soc. 2013, 135, 16758), supports the view that X contains a "diamond-core" Fe(III)/Fe(IV) center, with the irons bridged by two ligands. One bridging ligand is the oxo bridge (O_Br) derived from O₂ gas. Given the absence of a second inorganic oxygenic bridge, the second bridging ligand must be protein derived, and is most plausibly assigned as a carboxyl oxygen from E238.