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Issue 3, 2016
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Radical transfer in E. coli ribonucleotide reductase: a NH2Y731/R411A-α mutant unmasks a new conformation of the pathway residue 731

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Abstract

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides in all living organisms. The catalytic cycle of E. coli RNR involves a long-range proton-coupled electron transfer (PCET) from a tyrosyl radical (Y122˙) in subunit β2 to a cysteine (C439) in the active site of subunit α2, which subsequently initiates nucleotide reduction. This oxidation occurs over 35 Å and involves a specific pathway of redox active amino acids (Y122 ↔ [W48?] ↔ Y356 in β2 to Y731 ↔ Y730 ↔ C439 in α2). The mechanisms of the PCET steps at the interface of the α2β2 complex remain puzzling due to a lack of structural information for this region. Recently, DFT calculations on the 3-aminotyrosyl radical (NH2Y731˙)-α2 trapped by incubation of NH2Y731-α2/β2/CDP(substrate)/ATP(allosteric effector) suggested that R411-α2, a residue close to the α2β2 interface, interacts with NH2Y731˙ and accounts in part for its perturbed EPR parameters. To examine its role, we further modified NH2Y731-α2 with a R411A substitution. NH2Y731˙/R411A generated upon incubation of NH2Y731/R411A-α2/β2/CDP/ATP was investigated using multi-frequency (34, 94 and 263 GHz) EPR, 34 GHz pulsed electron–electron double resonance (PELDOR) and electron–nuclear double resonance (ENDOR) spectroscopies. The data indicate a large conformational change in NH2Y731˙/R411A relative to the NH2Y731˙ single mutant. Particularly, the inter-spin distance from NH2Y731˙/R411A in one αβ pair to Y122˙ in a second αβ pair decreases by 3 Å in the presence of the R411A mutation. This is the first experimental evidence for the flexibility of pathway residue Y731-α2 in an α2β2 complex and suggests a role for R411 in the stacked Y731/Y730 conformation involved in collinear PCET. Furthermore, NH2Y731˙/R411A serves as a probe of the PCET process across the subunit interface.

Graphical abstract: Radical transfer in E. coli ribonucleotide reductase: a NH2Y731/R411A-α mutant unmasks a new conformation of the pathway residue 731

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Article information


Submitted
14 Sep 2015
Accepted
06 Dec 2015
First published
09 Dec 2015

This article is Open Access
All publication charges for this article have been paid for by the Royal Society of Chemistry

Chem. Sci., 2016,7, 2170-2178
Article type
Edge Article
Author version available

Radical transfer in E. coli ribonucleotide reductase: a NH2Y731/R411A-α mutant unmasks a new conformation of the pathway residue 731

M. Kasanmascheff, W. Lee, T. U. Nick, J. Stubbe and M. Bennati, Chem. Sci., 2016, 7, 2170 DOI: 10.1039/C5SC03460D

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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