Spectroscopic and Electronic Structure Studies of Intermediate X in Ribonucleotide Reductase R2 and Two Variants: A Description of the FeIV-Oxo Bond in the FeIII−O−FeIV Dimer

Abstract

Spectroscopic and electronic structure studies of the class I Escherichia coli ribonucleotide reductase (RNR) intermediate X and three computationally derived model complexes are presented, compared, and evaluated to determine the electronic and geometric structure of the Fe^III−Fe^IV active site of intermediate X. Rapid freeze−quench (RFQ) EPR, absorption, and MCD were used to trap intermediate X in R2 wild-type (WT) and two variants, W48A and Y122F/Y356F. RFQ-EPR spin quantitation was used to determine the relative contributions of intermediate X and radicals present, while RFQ-MCD was used to specifically probe the Fe^III/Fe^IV active site, which displayed three Fe^IV d−d transitions between 16 700 and 22 600 cm^-1, two Fe^IV d−d spin-flip transitions between 23 500 and 24 300 cm^-1, and five oxo to Fe^IV and Fe^III charge transfer (CT) transitions between 25 000 and 32 000 cm^-1. The Fe^IV d−d transitions were perturbed in the two variants, confirming that all three d−d transitions derive from the d-π manifold. Furthermore, the Fe^IV d-π splittings in the WT are too large to correlate with a bis-μ-oxo structure. The assignment of the Fe^IV d−d transitions in WT intermediate X best correlates with a bridged μ-oxo/μ-hydroxo [Fe^III(μ-O)(μ-OH)Fe^IV] structure. The μ-oxo/μ-hydroxo core structure provides an important σ/π superexchange pathway, which is not present in the bis-μ-oxo structure, to promote facile electron transfer from Y122 to the remote Fe^IV through the bent oxo bridge, thereby generating the tyrosyl radical for catalysis.