Conformational Motions and Water Networks at the α/β Interface in E. coli Ribonucleotide Reductase

Abstract

Ribonucleotide reductases (RNRs) catalyze the conversion of all four ribonucleotides to deoxyribonucleotides and are essential for DNA synthesis in all organisms. The active form of E. coli Ia RNR is composed of two homodimers that form the active α2β2 complex. Catalysis is initiated by long-range radical translocation over a ∼32 Å proton-coupled electron transfer (PCET) pathway involving Y356β and Y731α at the interface. Resolving the PCET pathway at the α/β interface has been a long-standing challenge due to the lack of structural data. Herein, molecular dynamics simulations based on a recently solved cryogenic-electron microscopy structure of an active α2β2 complex are performed to examine the structure and fluctuations of interfacial water, as well as the hydrogen-bonding interactions and conformational motions of interfacial residues along the PCET pathway. Our free energy simulations reveal that Y731 is able to sample both a flipped-out conformation, where it points toward the interface to facilitate interfacial PCET with Y356, and a stacked conformation with Y730 to enable collinear PCET with this residue. Y356 and Y731 exhibit hydrogen-bonding interactions with interfacial water molecules and, in some conformations, share a bridging water molecule, suggesting that the primary proton acceptor for PCET from Y356 and from Y731 is interfacial water. The conformational flexibility of Y731 and the hydrogen-bonding interactions of both Y731 and Y356 with interfacial water and hydrogen-bonded water chains appear critical for effective radical translocation along the PCET pathway. These simulations are consistent with biochemical and spectroscopic data and provide previously unattainable atomic-level insights into the fundamental mechanism of RNR.

Concerted and Stepwise Proton-Coupled Electron Transfer for Tryptophan-Derivative Oxidation with Water as the Primary Proton Acceptor: Clarifying a Controversy

“… 44 Similar behavior has been suggested for Y at the interface between two subunits in Class 1 RNR. 45 , 46 CEPT from W with water as the primary proton acceptor, discussed in the Introduction section, is less viable due to the much larger p K a values of the reduced and oxidized forms. The large family of DNA photolyase and cryptochrome enzymes has a common three-W motif where one W is in a solvent-exposed position and has been shown to undergo ETPT.…”

Detection of Water Molecules on the Radical Transfer Pathway of Ribonucleotide Reductase by 17O Electron–Nuclear Double Resonance Spectroscopy

“…It might be caused by conformational distribution of this residue, which was found to have flexibility. 39 , 40 , 33 …”

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

“…The Y730/Y731 PCET reaction is indicated with purple square brackets. Figure adapted with permission from ref . Copyright 2020 American Chemical Society.…”

“…All of the simulations started from the cryo-EM structure of the α 2 β 2 complex (PDB ID: 6W4X), followed by immersion of the protein in a box of explicit water molecules. The system preparation and equilibration protocols were the same as those described in our previous work and are described in the Supporting Information (SI). Our previous simulations showed that the flipped-out conformer of Y731 is the thermodynamically favored conformation in the α subunit for this structure modeled as WT RNR with the AMBER ff14SB force field .…”

“…The system preparation and equilibration protocols were the same as those described in our previous work 30 and are described in the Supporting Information (SI). Our previous simulations 30 showed that the flipped-out conformer of Y731 is the thermodynamically favored conformation in the α subunit for this structure modeled as WT RNR with the AMBER ff14SB force field. 31 To model collinear PCET between Y730 and Y731, however, the stacked conformation is required.…”