Charge-Transfer Dynamics at the α/β Subunit Interface of a Photochemical Ribonucleotide Reductase

United States. National Institutes of Health (GM 29595

Charge Transfer Dynamics at the α/β Subunit Interface of a Photochemical Ribonucleotide Reductase

Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides to provide the monomeric building blocks for DNA replication and repair. Nucleotide reduction occurs by way of multi-step proton-coupled electron transfer (PCET) over a pathway of redox active amino acids spanning ~ 35 Å and two subunits (α2 and β2). Despite the fact that PCET in RNR is rapid, slow conformational changes mask kinetic examination of these steps. As such, we have pioneered methodology in which site-specific incorporation of a [ReI] photooxidant on the surface of the β2 subunit (photoβ2) allows photochemical oxidation of the adjacent PCET pathway residue β-Y356 and time-resolved spectroscopic observation of the ensuing reactivity. A series of photoβ2s capable of performing photoinitiated substrate turnover have been prepared in which four different fluorotyrosines (FnYs) are incorporated in place of β-Y356. The FnYs are deprotonated under biological conditions, undergo oxidation by electron transfer (ET) and provide a means by which to vary the ET driving force (ΔG°) with minimal additional perturbations across the series. We have used these features to map the correlation between ΔG° and kET both with and without the fully assembled photoRNR complex. The photooxidation of FnY356 within the α/β subunit interface occurs within the Marcus inverted region with a reorganization energy of λ ≈ 1 eV. We also observe enhanced electronic coupling between donor and acceptor (HDA) in the presence of an intact PCET pathway. Additionally, we have investigated the dynamics of proton transfer (PT) by a variety of methods including dependencies on solvent isotopic composition, buffer concentration, and pH. We present evidence for the role of α2 in facilitating PT during β-Y356 photooxidation; PT occurs by way of readily exchangeable positions and within a relatively “tight” subunit interface. These findings show that RNR controls ET by lowering λ, raising HDA, and directing PT both within and between individual polypeptide subunits.Chemistry and Chemical Biolog

Photochemical Generation of a Tryptophan Radical within the Subunit Interface of Ribonucleotide Reductase

United States. National Institutes of Health (GM029595

Modulation of Y356 Photooxidation in E. coli Class Ia Ribonucleotide Reductase by Y731 Across the α2:β2 Interface

Substrate turnover in class Ia ribonucleotide reductase (RNR) requires reversible radical transport across two subunits over 35 A, which occurs by a multi-step proton-coupled electron transfer mechanism. Using a photooxidant-labeled β2 subunit of Escherichia coli class Ia RNR, we demonstrate photoinitiated oxidation of a tyrosine in an α2:β2 complex, which results in substrate turnover. Using site-directed mutations of the redox-active tyrosines at the subunit interface—Y356F(β) and Y731F(α)—this oxidation is identified to be localized on Y356. The rate of Y356 oxidation depends on the presence of Y731 across the interface. This observation supports the proposal that unidirectional PCET across the Y356(β)–Y731(α)–Y730(α) triad is crucial to radical transport in RNR.Chemistry and Chemical Biolog

Patterns of oral anticoagulant use and outcomes in Asian patients with atrial fibrillation:a post-hoc analysis from the GLORIA-AF Registry

Background: Previous studies suggested potential ethnic differences in the management and outcomes of atrial fibrillation (AF). We aim to analyse oral anticoagulant (OAC) prescription, discontinuation, and risk of adverse outcomes in Asian patients with AF, using data from a global prospective cohort study. Methods: From the GLORIA-AF Registry Phase II–III (November 2011–December 2014 for Phase II, and January 2014–December 2016 for Phase III), we analysed patients according to their self-reported ethnicity (Asian vs. non-Asian), as well as according to Asian subgroups (Chinese, Japanese, Korean and other Asian). Logistic regression was used to analyse OAC prescription, while the risk of OAC discontinuation and adverse outcomes were analysed through Cox-regression model. Our primary outcome was the composite of all-cause death and major adverse cardiovascular events (MACE). The original studies were registered with ClinicalTrials.gov, NCT01468701, NCT01671007, and NCT01937377. Findings: 34,421 patients were included (70.0 ± 10.5 years, 45.1% females, 6900 (20.0%) Asian: 3829 (55.5%) Chinese, 814 (11.8%) Japanese, 1964 (28.5%) Korean and 293 (4.2%) other Asian). Most of the Asian patients were recruited in Asia (n = 6701, 97.1%), while non-Asian patients were mainly recruited in Europe (n = 15,449, 56.1%) and North America (n = 8378, 30.4%). Compared to non-Asian individuals, prescription of OAC and non-vitamin K antagonist oral anticoagulant (NOAC) was lower in Asian patients (Odds Ratio [OR] and 95% Confidence Intervals (CI): 0.23 [0.22–0.25] and 0.66 [0.61–0.71], respectively), but higher in the Japanese subgroup. Asian ethnicity was also associated with higher risk of OAC discontinuation (Hazard Ratio [HR] and [95% CI]: 1.79 [1.67–1.92]), and lower risk of the primary composite outcome (HR [95% CI]: 0.86 [0.76–0.96]). Among the exploratory secondary outcomes, Asian ethnicity was associated with higher risks of thromboembolism and intracranial haemorrhage, and lower risk of major bleeding. Interpretation: Our results showed that Asian patients with AF showed suboptimal thromboembolic risk management and a specific risk profile of adverse outcomes; these differences may also reflect differences in country-specific factors. Ensuring integrated and appropriate treatment of these patients is crucial to improve their prognosis. Funding: The GLORIA-AF Registry was funded by Boehringer Ingelheim GmbH.</p

Anticoagulant selection in relation to the SAMe-TT₂R₂ score in patients with atrial fibrillation:The GLORIA-AF registry

Aim: The SAMe-TT2R2 score helps identify patients with atrial fibrillation (AF) likely to have poor anticoagulation control during anticoagulation with vitamin K antagonists (VKA) and those with scores &gt;2 might be better managed with a target-specific oral anticoagulant (NOAC). We hypothesized that in clinical practice, VKAs may be prescribed less frequently to patients with AF and SAMe-TT2R2 scores &gt;2 than to patients with lower scores. Methods and results: We analyzed the Phase III dataset of the Global Registry on Long-Term Oral Antithrombotic Treatment in Patients with Atrial Fibrillation (GLORIA-AF), a large, global, prospective global registry of patients with newly diagnosed AF and ≥1 stroke risk factor. We compared baseline clinical characteristics and antithrombotic prescriptions to determine the probability of the VKA prescription among anticoagulated patients with the baseline SAMe-TT2R2 score &gt;2 and ≤ 2. Among 17,465 anticoagulated patients with AF, 4,828 (27.6%) patients were prescribed VKA and 12,637 (72.4%) patients an NOAC: 11,884 (68.0%) patients had SAMe-TT2R2 scores 0-2 and 5,581 (32.0%) patients had scores &gt;2. The proportion of patients prescribed VKA was 28.0% among patients with SAMe-TT2R2 scores &gt;2 and 27.5% in those with scores ≤2. Conclusions: The lack of a clear association between the SAMe-TT2R2 score and anticoagulant selection may be attributed to the relative efficacy and safety profiles between NOACs and VKAs as well as to the absence of trial evidence that an SAMe-TT2R2-guided strategy for the selection of the type of anticoagulation in NVAF patients has an impact on clinical outcomes of efficacy and safety. The latter hypothesis is currently being tested in a randomized controlled trial. Clinical trial registration: URL: https://www.clinicaltrials.gov//Unique identifier: NCT01937377, NCT01468701, and NCT01671007.</p

Kinetics and dynamics controlling proton-coupled electron transfer in ribonucleotide reductase

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2015.Cataloged from PDF version of thesis.Includes bibliographical references.Proton-coupled electron transfer (PCET) reactions comprise a fundamental mechanism for energy transduction in nature. In catalyzing the conversion of ribonucleotides to deoxyribonucleotides, ribonucleotide reductase (RNR) performs reversible, long-range PCET over a pathway of redox active amino acids ([beta]-Y₁₂₂ >////< [alpha]-C₄₃₉) that spans ~35 Å and two subunits. As such, RNR serves as a paradigm for the study of PCET in biology. Subunit interaction dynamics, examined by fluorescence spectroscopy, exposed mechanisms underlying allosteric control over PCET and contributed to an expanded kinetic model for turnover. Trapped meta-stable states of the active [alpha]₂[beta]₂ complex are dictated by the translocation of a single charge and attenuate dissociation 10⁴-fold. These trapped states were leveraged to resolve the stoichiometric distribution of the Y¹²²* cofactor from its ensemble average of 1.2 Y*/[beta]₂ , revealing that [beta]₂ contain either 2 or 0 Y*. Circumventing rate-limiting conformational changes that gate turnover, photoinitiated RNRs were prepared to allow photochemically driven Y₃₅₆ oxidation, and spectroscopic resolution of the ensuing reactivity. A series of photoRNRs containing unnatural FnYs (n = 2-3) and W in place of [beta]-Y₃₅₆ were prepared. All of these photo[beta]₂s give rise to transient absorption (TA) spectra consistent with their oxidized forms and undergo photochemically driven turnover. Time-resolved emission spectroscopy allowed examination of ET kinetics as a function of driving force within the [alpha]/[beta] subunit interface. Marcus-inverted kinetics were observed, providing reorganization and electronic coupling energies. Comparing ET and PCET kinetics as a function of pH, buffer concentration, oligomeric state, and buffer isotopic composition revealed new insights into biological control over PCET reactions and implicate a role of [alpha]₂ in facilitating proton transfer from [beta]-Y₃₅₆ Single wavelength TA kinetics provided direct measure of the rate constant for PCET through a, assignment of the rate-determining step as 3'-C-H bond cleavage by C₄₃₉ , and a lower bound of 7 for the associated 1° KIE. The pKa of proton acceptor(s) at the subunit interface, and the relative energies of individual radical intermediates were determined, revealing matched tuning to the surrounding environment and highlighting the subtlety of precision control underlying RNR catalysis.by Lisa Olshansky.Ph. D

Conformational Control over Proton-Coupled Electron Transfer in Metalloenzymes

From the reduction of dinitrogen to the oxidation of water, the chemical transformations catalyzed by metalloenzymes underlie global geo- and biochemical cycles. These reactions represent some of the most kinetically and thermodynamically challenging processes known. They require the complex choreography of nature’s fundamental building blocks: electrons and protons, to be carried out with utmost precision and accuracy; mistimed synchronicity can be fatal. Gated by macrostructural conformational changes, the rate-determining steps of catalysis in many of these enzymes consist of protein structural rearrangements. Accordingly, a pattern emerges in which it appears that nature has evolved to leverage changes in macromolecular protein structure to control changes in the metallocofactor microstructure. This critical review defines (where possible) and discusses the detailed molecular mechanisms of how metalloenzymes are able to efficiently convert allosteric binding energy into activation energy through conformational gating. Here, the proton-coupled electron transfer (PCET) mechanisms in biology stand as a paradigm for the interplay between molecular and electronic structural control. Taking nitrogenase, photosystem II, and ribonucleotide reductase as examples, we present the culmination of decades of study on each of these systems to clarify what is known regarding the interplay between structural changes and functional outcomes in these metalloenzyme linchpins

Conformational Control over Proton-Coupled Electron Transfer in Metalloenzymes

From the reduction of dinitrogen to the oxidation of water, the chemical transformations catalyzed by metalloenzymes underlie global geo- and biochemical cycles. These reactions represent some of the most kinetically and thermodynamically challenging processes known. They require the complex choreography of nature’s fundamental building blocks: electrons and protons, to be carried out with utmost precision and accuracy; mistimed synchronicity can be fatal. Gated by macrostructural conformational changes, the rate-determining steps of catalysis in many of these enzymes consist of protein structural rearrangements. Accordingly, a pattern emerges in which it appears that nature has evolved to leverage changes in macromolecular protein structure to control changes in the metallocofactor microstructure. This critical review defines (where possible) and discusses the detailed molecular mechanisms of how metalloenzymes are able to efficiently convert allosteric binding energy into activation energy through conformational gating. Here, the proton-coupled electron transfer (PCET) mechanisms in biology stand as a paradigm for the interplay between molecular and electronic structural control. Taking nitrogenase, photosystem II, and ribonucleotide reductase as examples, we present the culmination of decades of study on each of these systems to clarify what is known regarding the interplay between structural changes and functional outcomes in these metalloenzyme linchpins