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

Fabian Hecker,Joanne Stubbe,Marina Bennati

doi:10.1021/jacs.1c01359

Abstract

The role of water in biological proton-coupled electron transfer (PCET) is emerging as a key for understanding mechanistic details at atomic resolution. Here we demonstrate 17O high-frequency electron–nuclear double resonance (ENDOR) in conjunction with H217O-labeled protein buffer to establish the presence of ordered water molecules at three radical intermediates in an active enzyme complex, the α2β2E. coli ribonucleotide reductase. Our data give unambiguous evidence that all three, individually trapped, intermediates are hyperfine coupled to one water molecule with Tyr-O···17O distances in the range 2.8–3.1 Å. The availability of this structural information will allow for quantitative models of PCET in this prototype enzyme. The results also provide a spectroscopic signature for water H-bonded to a tyrosyl radical.

Highlights

The role of water in biological proton-coupled electron transfer (PCET) is emerging as a key for understanding mechanistic details at atomic resolution
Of particular interest is water involvement in electron transfer processes,[2−5] its action as a proton wire[6−8] or its role in proton-coupled electron transfer (PCET).[9−12] The identification of internal water in proteins can be achieved by X-ray diffraction.[13−15] the crystallization of transient protein complexes is difficult
One key approach for detection of water in biological systems has been the use of 17O-enriched water in conjunction with magnetic resonance spectroscopy.[16−21] Among these methods, electron paramagnetic resonance (EPR) can take advantage of high selectivity, as it detects nuclei only in the ligand sphere (r ≲ 1.5 nm)[22] of paramagnetic centers

Summary

Model of the

ARedox-active tyrosines 356, 731, and 730 are shown in cyan, electron transfer steps as red arrows, and proton transfer steps as blue arrows. We began with a DFT-optimized small model (25 atoms, details in section SI1) of Y356, as previous ENDOR spectra revealed 1H couplings consistent with one water at the H-bond distance rO−H ≈ 1.8 Å.31. For α2β2-NH2Y731, large-scale (215 atoms) DFT calculations previously proposed three models of the trapped intermediate (section SI10) Among these models, only one (model 3, Figure S15) contained a water molecule at an Hbond distance. The spectroscopic approach led to the first detection of ordered water molecules at three trapped radicals proposed to be representative of Y intermediates in the PCET of E. coli RNR These results verify previous hypotheses on the presence and role of water in the RNR mechanism and provide a new starting point for computational studies.

■ ACKNOWLEDGMENTS

Findings

■ REFERENCES