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 <b>g</b> Tensor

The radical concentrations and <i>g</i> factors of stable organic radicals in different lignin preparations were determined by X-band EPR at 9 GHz. We observed that the <i>g</i> 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 <i>g</i> 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 <i>g</i>_<i>xx</i>, <i>g</i>_<i>yy</i>, and <i>g</i>_<i>zz</i> components of the <b>g</b> 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 <i>o</i>-semiquinone radicals and can exist in different protonation states <b>SH3+</b>, <b>SH2</b>, <b>SH1-</b>, and <b>S2-</b>. The proposed model structures are supported by DFT calculations. The <i>g</i> principal values of the proposed structure were all in reasonable agreement with the experiments

ENDOR Spectroscopy and DFT Calculations: Evidence for the Hydrogen-Bond Network Within α2 in the PCET of E. coli Ribonucleotide Reductase

Escherichia coli class I ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides and is composed of two subunits: α2 and β2. β2 contains a stable di-iron tyrosyl radical (Y₁₂₂^•) cofactor required to generate a thiyl radical (C₄₃₉^•) in α2 over a distance of 35 Å, which in turn initiates the chemistry of the reduction process. The radical transfer process is proposed to occur by proton-coupled electron transfer (PCET) via a specific pathway: Y₁₂₂ ⇆ W₄₈[?] ⇆ Y₃₅₆ in β2, across the subunit interface to Y₇₃₁ ⇆ Y₇₃₀ ⇆ C₄₃₉ in α2. Within α2 a colinear PCET model has been proposed. To obtain evidence for this model, 3-amino tyrosine (NH₂Y) replaced Y₇₃₀ in α2, and this mutant was incubated with β2, cytidine 5′-diphosphate, and adenosine 5′-triphosphate to generate a NH₂Y₇₃₀^• in D₂O. [²H]-Electron–nuclear double resonance (ENDOR) spectra at 94 GHz of this intermediate were obtained, and together with DFT models of α2 and quantum chemical calculations allowed assignment of the prominent ENDOR features to two hydrogen bonds likely associated with C₄₃₉ and Y₇₃₁. A third proton was assigned to a water molecule in close proximity (2.2 Å O–H···O distance) to residue 730. The calculations also suggest that the unusual <i>g</i>-values measured for NH₂Y₇₃₀^• are consistent with the combined effect of the hydrogen bonds to Cys₄₃₉ and Tyr₇₃₁, both nearly perpendicular to the ring plane of NH₂Y_730. The results provide the first experimental evidence for the hydrogen-bond network between the pathway residues in α2 of the active RNR complex, for which no structural data are available

Structural Examination of the Transient 3-Aminotyrosyl Radical on the PCET Pathway of <i>E. coli</i> Ribonucleotide Reductase by Multifrequency EPR Spectroscopy

<i>E. coli</i> ribonucleotide reductase (RNR) catalyzes the conversion of nucleotides to deoxynucleotides, a process that requires long-range radical transfer over 35 Å from a tyrosyl radical (Y₁₂₂•) within the β2 subunit to a cysteine residue (C₄₃₉) within the α2 subunit. The radical transfer step is proposed to occur by proton-coupled electron transfer via a specific pathway consisting of Y₁₂₂ → W₄₈ → Y₃₅₆ in β2, across the subunit interface to Y₇₃₁ → Y₇₃₀ → C₄₃₉ in α2. Using the suppressor tRNA/aminoacyl-tRNA synthetase (RS) methodology, 3-aminotyrosine has been incorporated into position 730 in α2. Incubation of this mutant with β2, substrate, and allosteric effector resulted in loss of the Y₁₂₂• and formation of a new radical, previously proposed to be a 3-aminotyrosyl radical (NH₂Y•). In the current study [¹⁵N]- and [¹⁴N]-NH₂Y₇₃₀• have been generated in H₂O and D₂O and characterized by continuous wave 9 GHz EPR and pulsed EPR spectroscopies at 9, 94, and 180 GHz. The data give insight into the electronic and molecular structure of NH₂Y₇₃₀•. The <i>g</i> tensor (<i>g</i>_<i>x</i> = 2.0052, <i>g</i>_<i>y</i> = 2.0042, <i>g</i>_<i>z</i> = 2.0022), the orientation of the β-protons, the hybridization of the amine nitrogen, and the orientation of the amino protons relative to the plane of the aromatic ring were determined. The hyperfine coupling constants and geometry of the NH₂ moiety are consistent with an intramolecular hydrogen bond within NH₂Y₇₃₀•. This analysis is an essential first step in using the detailed structure of NH₂Y₇₃₀• to formulate a model for a PCET mechanism within α2 and for use of NH₂Y in other systems where transient Y•s participate in catalysis

Hydrogen Bond Network between Amino Acid Radical Intermediates on the Proton-Coupled Electron Transfer Pathway of <i>E. coli</i> 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•, <i>Escherichia coli</i>) in subunit β 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 β 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-β, Y₇₃₁- and Y₇₃₀-α, and each protein was incubated with the appropriate second subunit β­(α), CDP and effector ATP to trap an amino tyrosyl radical (NH₂Y•) in the active α2β2 complex. High-frequency (263 GHz) pulse electron paramagnetic resonance (EPR) of the NH₂Y•s reported the <i>g</i>_<i>x</i> values with unprecedented resolution and revealed strong electrostatic effects caused by the protein environment. ²H 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

Properties of Site-Specifically Incorporated 3‑Aminotyrosine in Proteins To Study Redox-Active Tyrosines: <i>Escherichia coli</i> Ribonucleotide Reductase as a Paradigm

3-Aminotyrosine (NH₂Y) has been a useful probe to study the role of redox active tyrosines in enzymes. This report describes properties of NH₂Y of key importance for its application in mechanistic studies. By combining the tRNA/NH₂Y-RS suppression technology with a model protein tailored for amino acid redox studies (α₃X, X = NH₂Y), the formal reduction potential of NH₂Y₃₂(O^•/OH) (<i><i>E</i>°′</i> = 395 ± 7 mV at pH 7.08 ± 0.05) could be determined using protein film voltammetry. We find that the Δ<i><i>E</i>°′</i> between NH₂Y₃₂(O^•/OH) and Y₃₂(O^•/OH) when measured under reversible conditions is ∼300–400 mV larger than earlier estimates based on irreversible voltammograms obtained on aqueous NH₂Y and Y. We have also generated D₆-NH₂Y₇₃₁-α2 of ribonucleotide reductase (RNR), which when incubated with β2/CDP/ATP generates the D₆-NH₂Y₇₃₁^•-α2/β2 complex. By multifrequency electron paramagnetic resonance (35, 94, and 263 GHz) and 34 GHz ¹H ENDOR spectroscopies, we determined the hyperfine coupling (hfc) constants of the amino protons that establish RNH₂^• planarity and thus minimal perturbation of the reduction potential by the protein environment. The amount of Y in the isolated NH₂Y-RNR incorporated by infidelity of the tRNA/NH₂Y-RS pair was determined by a generally useful LC-MS method. This information is essential to the utility of this NH₂Y probe to study any protein of interest and is employed to address our previously reported activity associated with NH₂Y-substituted RNRs

Structure of the Nucleotide Radical Formed during Reaction of CDP/TTP with the E441Q-α2β2 of <i>E. coli</i> Ribonucleotide Reductase

The <i>Escherichia coli</i> ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleotides and requires a diferric-tyrosyl radical cofactor for catalysis. RNR is composed of a 1:1 complex of two homodimeric subunits: α and β. Incubation of the E441Q-α mutant RNR with substrate CDP and allosteric effector TTP results in loss of the tyrosyl radical and formation of two new radicals on the 200 ms to min time scale. The first radical was previously established by stopped flow UV/vis spectroscopy and pulsed high field EPR spectroscopy to be a disulfide radical anion. The second radical was proposed to be a 4′-radical of a 3′-keto-2′-deoxycytidine 5′-diphosphate. To identify the structure of the nucleotide radical [1′-²H], [2′-²H], [4′-²H], [5′-²H], [U−¹³C, ¹⁵N], [U−¹⁵N], and [5,6 -²H] CDP and [β-²H] cysteine-α were synthesized and incubated with E441Q-α2β2 and TTP. The nucleotide radical was examined by 9 GHz and 140 GHz pulsed EPR spectroscopy and 35 GHz ENDOR spectroscopy. Substitution of ²H at C4′ and C1′ altered the observed hyperfine interactions of the nucleotide radical and established that the observed structure was not that predicted. DFT calculations (B3LYP/IGLO-III/B3LYP/TZVP) were carried out in an effort to recapitulate the spectroscopic observations and lead to a new structure consistent with all of the experimental data. The results indicate, unexpectedly, that the radical is a semidione nucleotide radical of cytidine 5′-diphosphate. The relationship of this radical to the disulfide radical anion is discussed