Glutamate Mediates Proton-Coupled Electron Transfer Between Tyrosines 730 and 731 in Escherichia coli Ribonucleotide Reductase

Ribonucleotide reductase (RNR) is
an essential enzyme in DNA synthesis
for all living organisms. It reduces ribonucleotides to the corresponding
deoxyribonucleotides by a reversible radical transfer mechanism. The
active form of E. coli Ia RNR is composed
of two subunits, α and β, which form an active asymmetric
α2β2 complex. The radical transfer
pathway involves a series of proton-coupled electron transfer (PCET)
reactions spanning α and β over ∼32 Å. Herein,
quantum mechanical/molecular mechanical free energy simulations of
PCET between tyrosine residues Y730 and Y731 are performed on the
recently solved cryo-EM structure of the active α2β2 complex, which includes a pre-turnover α/β
pair with an ordered PCET pathway and a post-turnover α′/β′
pair. The free energy surfaces in both the pre- and post-turnover
states are computed. According to the simulations, forward radical
transfer from Y731 to Y730 is thermodynamically favored in the pre-turnover
state, and backward radical transfer is favored in the post-turnover
state, consistent with the reversible mechanism. E623, a glutamate
residue that is near these tyrosines only in the pre-turnover state,
is discovered to play a key role in facilitating forward radical transfer
by thermodynamically stabilizing the radical on Y730 through hydrogen-bonding
and electrostatic interactions and lowering the free energy barrier
via a proton relay mechanism. Introduction of fluorinated Y731 exhibits
expected thermodynamic trends without altering the basic mechanism.
These simulations suggest that E623 influences the directionality
of PCET between Y731 and Y730 and predict that mutation of E623 will
impact catalysis.