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  19F Electron-nuclear double resonance reveals interaction between redox-active tyrosines across the α/β interface of E. coli ribonucleotide reductase

Meyer, A., Kehl, A., Cui, C., Reichardt, F. A. K., Hecker, F., Funk, L.-M., et al. (2022). 19F Electron-nuclear double resonance reveals interaction between redox-active tyrosines across the α/β interface of E. coli ribonucleotide reductase. Journal of the American Chemical Society, 144, 11270-11282. doi:10.1021/jacs.2c02906.

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 Creators:
Meyer, A.1, Author           
Kehl, A.1, Author           
Cui, C., Author
Reichardt, F. A. K.1, Author           
Hecker, F.1, Author           
Funk, L.-M.2, Author           
Ghosh, M. K., Author
Pan, K. T.3, Author           
Urlaub, H.3, Author           
Tittmann, K.2, Author           
Stubbe, J., Author
Bennati, M.1, Author           
Affiliations:
1Research Group of Electron Paramagnetic Resonance, Max Planck Institute for Multidisciplinary Sciences, Max Planck Society, ou_3350281              
2Department of Structural Dynamics, Max Planck Institute for Multidisciplinary Sciences, Max Planck Society, ou_3350272              
3Research Group of Bioanalytical Mass Spectrometry, Max Planck Institute for Multidisciplinary Sciences, Max Planck Society, ou_3350290              

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Free keywords: Chemical structure; Conformation; Coupling reactions; Electron paramagnetic resonance spectroscopy; Organic compounds;
 Abstract: Ribonucleotide reductases (RNRs) catalyze the reduction of ribonucleotides to deoxyribonucleotides, thereby playing a key role in DNA replication and repair. Escherichia coli class Ia RNR is an α2β2 enzyme complex that uses a reversible multistep radical transfer (RT) over 32 Å across its two subunits, α and β, to initiate, using its metallo-cofactor in β2, nucleotide reduction in α2. Each step is proposed to involve a distinct proton-coupled electron-transfer (PCET) process. An unresolved step is the RT involving Y356(β) and Y731(α) across the α/β interface. Using 2,3,5-F3Y122-β2 with 3,5-F2Y731-α2, GDP (substrate) and TTP (allosteric effector), a Y356• intermediate was trapped and its identity was verified by 263 GHz electron paramagnetic resonance (EPR) and 34 GHz pulse electron–electron double resonance spectroscopies. 94 GHz 19F electron-nuclear double resonance spectroscopy allowed measuring the interspin distances between Y356• and the 19F nuclei of 3,5-F2Y731 in this RNR mutant. Similar experiments with the double mutant E52Q/F3Y122-β2 were carried out for comparison to the recently published cryo-EM structure of a holo RNR complex. For both mutant combinations, the distance measurements reveal two conformations of 3,5-F2Y731. Remarkably, one conformation is consistent with 3,5-F2Y731 within the H-bond distance to Y356•, whereas the second one is consistent with the conformation observed in the cryo-EM structure. The observations unexpectedly suggest the possibility of a colinear PCET, in which electron and proton are transferred from the same donor to the same acceptor between Y356 and Y731. The results highlight the important role of state-of-the-art EPR spectroscopy to decipher this mechanism.

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Language(s): eng - English
 Dates: 2022-06-022022
 Publication Status: Published in print
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 Rev. Type: -
 Identifiers: DOI: 10.1021/jacs.2c02906
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Project name : BIO-enMR
Grant ID : 101020262
Funding program : Horizon 2020 (H2020)
Funding organization : European Commission (EC)
Project name : -
Grant ID : BE 1680/7-2
Funding program : -
Funding organization : German Research Foundation (DFG)

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Title: Journal of the American Chemical Society
Source Genre: Journal
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Pages: - Volume / Issue: 144 Sequence Number: - Start / End Page: 11270 - 11282 Identifier: ISSN: 0002-7863
ISSN: 1520-5126