Spectroscopic Evidence for a H Bond Network at Y356 Located at the Subunit Interface of Active E. coli Ribonucleotide Reductase.

The reaction catalyzed by E. coli ribonucleotide reductase (RNR) composed of α and ß subunits that form an active α2ß2 complex is a paradigm for proton-coupled electron transfer (PCET) processes in biological transformations. ß2 contains the diferric tyrosyl radical (Y122·) cofactor that initiates radical transfer (RT) over 35 Å via a specific pathway of amino acids (Y122· ⇆ [W48] ⇆ Y356 in ß2 to Y731 ⇆ Y730 ⇆ C439 in α2). Experimental evidence exists for colinear and orthogonal PCET in α2 and ß2, respectively. No mechanistic model yet exists for the PCET across the subunit (α/ß) interface. Here, we report unique EPR spectroscopic features of Y356·-ß, the pathway intermediate generated by the reaction of 2,3,5-F3Y122·-ß2/CDP/ATP with wt-α2, Y731F-α2, or Y730F-α2. High field EPR (94 and 263 GHz) reveals a dramatically perturbed g tensor. [1H] and [2H]-ENDOR reveal two exchangeable H bonds to Y356·: a moderate one almost in-plane with the π-system and a weak one. DFT calculation on small models of Y· indicates that two in-plane, moderate H bonds (rO-H ∼1.8-1.9 Å) are required to reproduce the gx value of Y356· (wt-α2). The results are consistent with a model, in which a cluster of two, almost symmetrically oriented, water molecules provide the two moderate H bonds to Y356· that likely form a hydrogen bond network of water molecules involved in either the reversible PCET across the subunit interface or in H+ release to the solvent during Y356 oxidation.

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

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 ß2 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 ß2 to Y731 ↔ Y730 ↔ C439 in α2). The mechanisms of the PCET steps at the interface of the α2ß2 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/ß2/CDP(substrate)/ATP(allosteric effector) suggested that R411-α2, a residue close to the α2ß2 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/ß2/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 αß pair to Y122˙ in a second αß 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 α2ß2 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.

High-Field Electron Paramagnetic Resonance and Density Functional Theory Study of Stable Organic Radicals in Lignin: Influence of the Extraction Process, Botanical Origin, and Protonation Reactions on the Radical g Tensor.

The radical concentrations and g factors of stable organic radicals in different lignin preparations were determined by X-band EPR at 9 GHz. We observed that the g factors of these radicals are largely determined by the extraction process and not by the botanical origin of the lignin. The parameter mostly influencing the g factor is the pH value during lignin extraction. This effect was studied in depth using high-field EPR spectroscopy at 263 GHz. We were able to determine the gxx, gyy, and gzz components of the g tensor of the stable organic radicals in lignin. With the enhanced resolution of high-field EPR, distinct radical species could be found in this complex polymer. The radical species are assigned to substituted o-semiquinone radicals and can exist in different protonation states SH3+, SH2, SH1-, and S2-. The proposed model structures are supported by DFT calculations. The g principal values of the proposed structure were all in reasonable agreement with the experiments.

Hydrogen bond network between amino acid radical intermediates on the proton-coupled electron transfer pathway of E. coli α2 ribonucleotide reductase.

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides in all organisms. In all Class Ia RNRs, initiation of nucleotide diphosphate (NDP) reduction requires a reversible oxidation over 35 Å by a tyrosyl radical (Y122•, Escherichia coli) in subunit ß of a cysteine (C439) in the active site of subunit α. This radical transfer (RT) occurs by a specific pathway involving redox active tyrosines (Y122 ⇆ Y356 in ß to Y731 ⇆ Y730 ⇆ C439 in α); each oxidation necessitates loss of a proton coupled to loss of an electron (PCET). To study these steps, 3-aminotyrosine was site-specifically incorporated in place of Y356-ß, Y731- and Y730-α, and each protein was incubated with the appropriate second subunit ß(α), CDP and effector ATP to trap an amino tyrosyl radical (NH2Y•) in the active α2ß2 complex. High-frequency (263 GHz) pulse electron paramagnetic resonance (EPR) of the NH2Y•s reported the gx values with unprecedented resolution and revealed strong electrostatic effects caused by the protein environment. (2)H electron-nuclear double resonance (ENDOR) spectroscopy accompanied by quantum chemical calculations provided spectroscopic evidence for hydrogen bond interactions at the radical sites, i.e., two exchangeable H bonds to NH2Y730•, one to NH2Y731• and none to NH2Y356•. Similar experiments with double mutants α-NH2Y730/C439A and α-NH2Y731/Y730F allowed assignment of the H bonding partner(s) to a pathway residue(s) providing direct evidence for colinear PCET within α. The implications of these observations for the PCET process within α and at the interface are discussed.