Predicting monoclonal antibody binding sequences from a sparse sampling of all possible sequences.

Previous work has shown that binding of target proteins to a sparse, unbiased sample of all possible peptide sequences is sufficient to train a machine learning model that can then predict, with statistically high accuracy, target binding to any possible peptide sequence of similar length. Here, highly sequence-specific molecular recognition is explored by measuring binding of 8 monoclonal antibodies (mAbs) with specific linear cognate epitopes to an array containing 121,715 near-random sequences about 10 residues in length. Network models trained on resulting sequence-binding values are used to predict the binding of each mAb to its cognate sequence and to an in silico generated one million random sequences. The model always ranks the binding of the cognate sequence in the top 100 sequences, and for 6 of the 8 mAbs, the cognate sequence ranks in the top ten. Practically, this approach has potential utility in selecting highly specific mAbs for therapeutics or diagnostics. More fundamentally, this demonstrates that very sparse random sampling of a large amino acid sequence spaces is sufficient to generate comprehensive models predictive of highly specific molecular recognition.

Modeling the sequence dependence of differential antibody binding in the immune response to infectious disease.

Past studies have shown that incubation of human serum samples on high density peptide arrays followed by measurement of total antibody bound to each peptide sequence allows detection and discrimination of humoral immune responses to a variety of infectious diseases. This is true even though these arrays consist of peptides with near-random amino acid sequences that were not designed to mimic biological antigens. This "immunosignature" approach, is based on a statistical evaluation of the binding pattern for each sample but it ignores the information contained in the amino acid sequences that the antibodies are binding to. Here, similar array-based antibody profiles are instead used to train a neural network to model the sequence dependence of molecular recognition involved in the immune response of each sample. The binding profiles used resulted from incubating serum from 5 infectious disease cohorts (Hepatitis B and C, Dengue Fever, West Nile Virus and Chagas disease) and an uninfected cohort with 122,926 peptide sequences on an array. These sequences were selected quasi-randomly to represent an even but sparse sample of the entire possible combinatorial sequence space (~1012). This very sparse sampling of combinatorial sequence space was sufficient to capture a statistically accurate representation of the humoral immune response across the entire space. Processing array data using the neural network not only captures the disease-specific sequence-binding information but aggregates binding information with respect to sequence, removing sequence-independent noise and improving the accuracy of array-based classification of disease compared with the raw binding data. Because the neural network model is trained on all samples simultaneously, a highly condensed representation of the differential information between samples resides in the output layer of the model, and the column vectors from this layer can be used to represent each sample for classification or unsupervised clustering applications.

Structure and biological evaluation of Caenorhabditis elegans CISD-1/mitoNEET, a KLP-17 tail domain homologue, supports attenuation of paraquat-induced oxidative stress through a p38 MAPK-mediated antioxidant defense response.

CISD-1/mitoNEET is an evolutionarily conserved outer mitochondrial membrane [2Fe-2S] protein that regulates mitochondrial function and morphology. The [2Fe-2S] clusters are redox reactive and shown to mediate oxidative stress in vitro and in vivo. However, there is limited research studying CISD-1/mitoNEET mediation of oxidative stress in response to environmental stressors. In this study, we have determined the X-ray crystal structure of Caenorhabditis elegans CISD-1/mitoNEET homologue and evaluated the mechanisms of oxidative stress resistance to the pro-oxidant paraquat in age-synchronized populations by generating C. elegans gain and loss of function CISD-1 models. The structure of the C. elegans CISD-1/mitoNEET soluble domain refined at 1.70-Å resolution uniquely shows a reversible disulfide linkage at the homo-dimeric interface and also represents the N-terminal tail domain for dimerization of the cognate kinesin motor protein KLP-17 involved in chromosome segregation dynamics and germline development of the nematode. Moreover, overexpression of CISD-1/mitoNEET in C. elegans has revealed beneficial effects on oxidative stress resistance against paraquat-induced reactive oxygen species generation, corroborated by increased activation of the p38 mitogen-activated protein kinase (MAPK) signaling cascade.

Influence of Polar Mutations on the Electronic and Structural Properties of QA-· in Bacterial Reaction Centers.

Reaction centers from Rhodobacter sphaeroides with residue M265 mutated from isoleucine to threonine, serine, and asparagine (M265IT, M265IS, and M265IN, respectively) in the QA-· state are studied by high-resolution electron spin echo envelope modulation (ESEEM) and electron nuclear double resonance spectroscopy methods to investigate the structural characteristics of these mutants influencing the redox properties of the QA site. All three mutants decrease the redox midpoint potential (Em) of QA by ∼0.1 V, yet the mechanism for this drop in Em is unclear. In this work, we examine (i) the hydrogen bonding interactions between QA-· and residues histidine M219 and alanine M260, (ii) the electron spin density distribution of the semiquinone, and (iii) the orientations of the ubiquinone methoxy substituents. 13C measurements show no significant contribution of methoxy dihedral angles to the observed decrease in Em for the QA mutants. Instead, 14N three-pulse ESEEM data suggest that electrostatic or hydrogen bond formation between the mutated M265 side chain and His-M219 Nδ may be involved in the observed lowering of the QA midpoint potential. For mutant M265IN, analysis of the proton hyperfine couplings reveals a weakened hydrogen bond network, resulting in an altered QA-· spin density distribution. The magnetic resonance study presented here is most consistent with an electrostatic or structural perturbation of the His-M219 Nδ hydrogen bond in these mutants as a mechanism for the ∼0.1 V decrease in QA Em.

Escherichia coli amino acid auxotrophic expression host strains for investigating protein structure-function relationships.

A set of C43(DE3) and BL21(DE3) Escherichia coli host strains that are auxotrophic for various amino acids is briefly reviewed. These strains require the addition of a defined set of one or more amino acids in the growth medium, and have been specifically designed for overproduction of membrane or water-soluble proteins selectively labelled with stable isotopes, such as 2H, 13C and 15N. The strains described here are available for use and have been deposited into public strain banks. Although they cannot fully eliminate the possibility of isotope dilution and mixing, metabolic scrambling of the different amino acid types can be minimized through a careful consideration of the bacterial metabolic pathways. The use of a suitable auxotrophic expression host strain with an appropriately isotopically labelled growth medium ensures high levels of isotope labelling efficiency as well as selectivity for providing deeper insight into protein structure-function relationships.

Comprehensive Prediction of Molecular Recognition in a Combinatorial Chemical Space Using Machine Learning.

In combinatorial chemical approaches, optimizing the composition and arrangement of building blocks toward a particular function has been done using a number of methods, including high throughput molecular screening, molecular evolution, and computational prescreening. Here, a different approach is considered that uses sparse measurements of library molecules as the input to a machine learning algorithm which generates a comprehensive, quantitative relationship between covalent molecular structure and function that can then be used to predict the function of any molecule in the possible combinatorial space. To test the feasibility of the approach, a defined combinatorial chemical space consisting of ∼1012 possible linear combinations of 16 different amino acids was used. The binding of a very sparse, but nearly random, sampling of this amino acid sequence space to 9 different protein targets is measured and used to generate a general relationship between peptide sequence and binding for each target. Surprisingly, measuring as little as a few hundred to a few thousand of the ∼1012 possible molecules provides sufficient training to be highly predictive of the binding of the remaining molecules in the combinatorial space. Furthermore, measuring only amino acid sequences that bind weakly to a target allows the accurate prediction of which sequences will bind 10-100 times more strongly. Thus, the molecular recognition information contained in a tiny fraction of molecules in this combinatorial space is sufficient to characterize any set of molecules randomly selected from the entire space, a fact that potentially has significant implications for the design of new chemical function using combinatorial chemical libraries.

Structure of a trapped radical transfer pathway within a ribonucleotide reductase holocomplex.

Ribonucleotide reductases (RNRs) are a diverse family of enzymes that are alone capable of generating 2'-deoxynucleotides de novo and are thus critical in DNA biosynthesis and repair. The nucleotide reduction reaction in all RNRs requires the generation of a transient active site thiyl radical, and in class I RNRs, this process involves a long-range radical transfer between two subunits, α and ß. Because of the transient subunit association, an atomic resolution structure of an active α2ß2 RNR complex has been elusive. We used a doubly substituted ß2, E52Q/(2,3,5)-trifluorotyrosine122-ß2, to trap wild-type α2 in a long-lived α2ß2 complex. We report the structure of this complex by means of cryo-electron microscopy to 3.6-angstrom resolution, allowing for structural visualization of a 32-angstrom-long radical transfer pathway that affords RNR activity.

Convolutional Neural Network Analysis of Two-Dimensional Hyperfine Sublevel Correlation Electron Paramagnetic Resonance Spectra.

A machine learning approach is presented for analyzing complex two-dimensional hyperfine sublevel correlation electron paramagnetic resonance (HYSCORE EPR) spectra with the proficiency of an expert spectroscopist. The computer vision algorithm requires no training on experimental data; rather, all of the spin physics required to interpret the spectra are learned from simulations alone. This approach is therefore applicable even when insufficient experimental data exist to train the algorithm. The neural network is demonstrated to be capable of utilizing the full information content of two-dimensional 14N HYSCORE spectra to predict the magnetic coupling parameters and their underlying probability distributions that were previously inaccessible. The predicted hyperfine ( a, T) and 14N quadrupole ( K, η) coupling constants deviate from the previous manual analyses of the experimental spectra on average by 0.11 MHz, 0.09 MHz, 0.19 MHz, and 0.09, respectively.

Metal-free class Ie ribonucleotide reductase from pathogens initiates catalysis with a tyrosine-derived dihydroxyphenylalanine radical.

All cells obtain 2'-deoxyribonucleotides for DNA synthesis through the activity of a ribonucleotide reductase (RNR). The class I RNRs found in humans and pathogenic bacteria differ in (i) use of Fe(II), Mn(II), or both for activation of the dinuclear-metallocofactor subunit, ß; (ii) reaction of the reduced dimetal center with dioxygen or superoxide for this activation; (iii) requirement (or lack thereof) for a flavoprotein activase, NrdI, to provide the superoxide from O2; and (iv) use of either a stable tyrosyl radical or a high-valent dimetal cluster to initiate each turnover by oxidizing a cysteine residue in the α subunit to a radical (Cys•). The use of manganese by bacterial class I, subclass b-d RNRs, which contrasts with the exclusive use of iron by the eukaryotic Ia enzymes, appears to be a countermeasure of certain pathogens against iron deprivation imposed by their hosts. Here, we report a metal-free type of class I RNR (subclass e) from two human pathogens. The Cys• in its α subunit is generated by a stable, tyrosine-derived dihydroxyphenylalanine radical (DOPA•) in ß. The three-electron oxidation producing DOPA• occurs in Escherichia coli only if the ß is coexpressed with the NrdI activase encoded adjacently in the pathogen genome. The independence of this new RNR from transition metals, or the requirement for a single metal ion only transiently for activation, may afford the pathogens an even more potent countermeasure against transition metal-directed innate immunity.

g-Tensor Directions in the Protein Structural Frame of Hyperthermophilic Archaeal Reduced Rieske-Type Ferredoxin Explored by 13C Pulsed Electron Paramagnetic Resonance.

Interpretation of magnetic resonance data in the context of structural and chemical biology requires prior knowledge of the g-tensor directions for paramagnetic metallo-cofactors with respect to the protein structural frame. Access to this information is often limited by the strict requirement of suitable protein crystals for single-crystal electron paramagnetic resonance (EPR) measurements or the reliance on protons (with ambiguous locations in crystal structures) near the paramagnetic metal site. Here we develop a novel pulsed EPR approach with selective 13Cß-cysteine labeling of model [2Fe-2S] proteins to help bypass these problems. Analysis of the 13Cß-cysteine hyperfine tensors reproduces the g-tensor of the Pseudomonas putida ISC-like [2Fe-2S] ferredoxin (FdxB). Its application to the hyperthermophilic archaeal Rieske-type [2Fe-2S] ferredoxin (ARF) from Sulfolobus solfataricus, for which the single-crystal EPR approach was not feasible, supports the best-fit g x-, g z-, and g y-tensor directions of the reduced cluster as nearly along Fe-Fe, S-S, and the cluster plane normal, respectively. These approximate principal directions of the reduced ARF g-tensor, explored by 13C pulsed EPR, are less skewed from the cluster molecular axes and are largely consistent with those previously determined by single-crystal EPR for the cytochrome bc1-associated, reduced Rieske [2Fe-2S] center. This suggests the approximate g-tensor directions are conserved across the phylogenetically and functionally divergent Rieske-type [2Fe-2S] proteins.

Two-Dimensional Pulsed EPR Resolves Hyperfine Coupling Strain in Nitrogen Hydrogen Bond Donors of Semiquinone Intermediates.

Hydrogen bonding between semiquinone (SQ) intermediates and side-chain or backbone nitrogens in protein quinone processing sites (Q-sites) is a common motif. Previous studies on SQs from multiple protein environments have reported specific features in the 15N HYSCORE spectra not reproducible by a theory based on fixed hyperfine parameters, and the source of these lineshape distortions remained unknown. In this work, using the spectra of the SQ in the Q-sites of wild-type and mutant D75H cytochrome bo3 ubiquinol oxidase from Escherichia coli, we have explained the observed additional features as originating from a-strain of the isotropic hyperfine coupling. In two-dimensional spectra, the a-strain manifests as well-resolved lineshape distortions of the basic cross-ridges and accompanying lines of low intensity in the opposite quadrant that allow its direct analysis. We have shown that their appearance is regulated by the relative values of the strain width, Δ a, and parameter, δ = |2 a + T| - 4ν15N. α-strain provides a direct measure of the structural dynamics and heterogeneity of the O···H···N bond in the SQ systems.

Unpaired Electron Spin Density Distribution across Reduced [2Fe-2S] Cluster Ligands by 13Cß-Cysteine Labeling.

Iron-sulfur clusters are one of the most versatile and ancient classes of redox mediators in biology. The roles that these metal centers take on are predominantly determined by the number and types of coordinating ligands (typically cysteine and histidine) that modify the electronic structure of the cluster. Here we map the spin density distribution onto the cysteine ligands for the three major classes of the protein-bound, reduced [2Fe-2S](His)n(Cys)4-n (n = 0, 1, 2) cluster by selective cysteine-13Cß isotope labeling. The spin distribution is highly asymmetric in all three systems and delocalizes further along the reduced Fe2+ ligands than the nonreducible Fe3+ ligands for all clusters studied. The preferential spin transfer onto the chemically reactive Fe2+ ligands is consistent with the structural concept that the orientation of the cluster in proteins is not arbitrarily decided, but rather is optimized such that it is likely to facilitate better electronic coupling with redox partners. The resolution of all cysteine-13Cß hyperfine couplings and their assignments provides a measure of the relative covalencies of the metal-thiolate bonds not readily available to other techniques.

Determination of the Complete Spin Density Distribution in 13C-Labeled Protein-Bound Radical Intermediates Using Advanced 2D Electron Paramagnetic Resonance Spectroscopy and Density Functional Theory.

Determining the complete electron spin density distribution for protein-bound radicals, even with advanced pulsed electron paramagnetic resonance (EPR) methods, is a formidable task. Here we present a strategy to overcome this problem combining multifrequency HYSCORE and ENDOR measurements on site-specifically 13C-labeled samples with DFT calculations on model systems. As a demonstration of this approach, pulsed EPR experiments are performed on the primary QA and secondary QB ubisemiquinones of the photosynthetic reaction center from Rhodobacter sphaeroides 13C-labeled at the ring and tail positions. Despite the large number of nuclei interacting with the unpaired electron in these samples, two-dimensional X- and Q-band HYSCORE and orientation selective Q-band ENDOR resolve and allow for a characterization of the eight expected 13C resonances from significantly different hyperfine tensors for both semiquinones. From these results we construct, for the first time, the most complete experimentally determined maps of the s- and pπ-orbital spin density distributions for any protein organic cofactor radical to date. This work lays a foundation for understanding the relationship between the electronic structure of semiquinones and their functional properties, and introduces new techniques for mapping out the spin density distribution that are readily applicable to other systems.

Conformationally Dynamic Radical Transfer within Ribonucleotide Reductase.

Ribonucleotide reductases (RNR) catalyze the reduction of nucleotides to deoxynucleotides through a mechanism involving an essential cysteine based thiyl radical. In the E. coli class 1a RNR the thiyl radical (C439•) is a transient species generated by radical transfer (RT) from a stable diferric-tyrosyl radical cofactor located >35 Å away across the α2:ß2 subunit interface. RT is facilitated by sequential proton-coupled electron transfer (PCET) steps along a pathway of redox active amino acids (Y122ß â†” [W48ß?] ↔ Y356ß â†” Y731α ↔ Y730α ↔ C439α). The mutant R411A(α) disrupts the H-bonding environment and conformation of Y731, ostensibly breaking the RT pathway in α2. However, the R411A protein retains significant enzymatic activity, suggesting Y731 is conformationally dynamic on the time scale of turnover. Installation of the radical trap 3-amino tyrosine (NH2Y) by amber codon suppression at positions Y731 or Y730 and investigation of the NH2Y• trapped state in the active α2:ß2 complex by HYSCORE spectroscopy validate that the perturbed conformation of Y731 in R411A-α2 is dynamic, reforming the H-bond between Y731 and Y730 to allow RT to propagate to Y730. Kinetic studies facilitated by photochemical radical generation reveal that Y731 changes conformation on the ns-µs time scale, significantly faster than the enzymatic kcat. Furthermore, the kinetics of RT across the subunit interface were directly assessed for the first time, demonstrating conformationally dependent RT rates that increase from 0.6 to 1.6 × 104 s-1 when comparing wild type to R411A-α2, respectively. These results illustrate the role of conformational flexibility in modulating RT kinetics by targeting the PCET pathway of radical transport.

Glutamate 52-ß at the α/ß subunit interface of Escherichia coli class Ia ribonucleotide reductase is essential for conformational gating of radical transfer.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleoside diphosphate substrates (S) to deoxynucleotides with allosteric effectors (e) controlling their relative ratios and amounts, crucial for fidelity of DNA replication and repair. Escherichia coli class Ia RNR is composed of α and ß subunits that form a transient, active α2ß2 complex. The E. coli RNR is rate-limited by S/e-dependent conformational change(s) that trigger the radical initiation step through a pathway of 35 Å across the subunit (α/ß) interface. The weak subunit affinity and complex nucleotide-dependent quaternary structures have precluded a molecular understanding of the kinetic gating mechanism(s) of the RNR machinery. Using a docking model of α2ß2 created from X-ray structures of α and ß and conserved residues from a new subclassification of the E. coli Ia RNR (Iag), we identified and investigated four residues at the α/ß interface (Glu350 and Glu52 in ß2 and Arg329 and Arg639 in α2) of potential interest in kinetic gating. Mutation of each residue resulted in loss of activity and with the exception of E52Q-ß2, weakened subunit affinity. An RNR mutant with 2,3,5-trifluorotyrosine radical (F3Y122•) replacing the stable Tyr122• in WT-ß2, a mutation that partly overcomes conformational gating, was placed in the E52Q background. Incubation of this double mutant with His6-α2/S/e resulted in an RNR capable of catalyzing pathway-radical formation (Tyr356•-ß2), 0.5 eq of dCDP/F3Y122•, and formation of an α2ß2 complex that is isolable in pulldown assays over 2 h. Negative stain EM images with S/e (GDP/TTP) revealed the uniformity of the α2ß2 complex formed.

Formal Reduction Potentials of Difluorotyrosine and Trifluorotyrosine Protein Residues: Defining the Thermodynamics of Multistep Radical Transfer.

Redox-active tyrosines (Ys) play essential roles in enzymes involved in primary metabolism including energy transduction and deoxynucleotide production catalyzed by ribonucleotide reductases (RNRs). Thermodynamic characterization of Ys in solution and in proteins remains a challenge due to the high reduction potentials involved and the reactive nature of the radical state. The structurally characterized α3Y model protein has allowed the first determination of formal reduction potentials (E°') for a Y residing within a protein (Berry, B. W.; Martínez-Rivera, M. C.; Tommos, C. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 9739-9743). Using Schultz's technology, a series of fluorotyrosines (FnY, n = 2 or 3) was site-specifically incorporated into α3Y. The global protein properties of the resulting α3(3,5)F2Y, α3(2,3,5)F3Y, α3(2,3)F2Y and α3(2,3,6)F3Y variants are essentially identical to those of α3Y. A protein film square-wave voltammetry approach was developed to successfully obtain reversible voltammograms and E°'s of the very high-potential α3FnY proteins. E°'(pH 5.5; α3FnY(O•/OH)) spans a range of 1040 ± 3 mV to 1200 ± 3 mV versus the normal hydrogen electrode. This is comparable to the potentials of the most oxidizing redox cofactors in nature. The FnY analogues, and the ability to site-specifically incorporate them into any protein of interest, provide new tools for mechanistic studies on redox-active Ys in proteins and on functional and aberrant hole-transfer reactions in metallo-enzymes. The former application is illustrated here by using the determined α3FnY ΔE°'s to model the thermodynamics of radical-transfer reactions in FnY-RNRs and to experimentally test and support the key prediction made.

Glutamate 350 Plays an Essential Role in Conformational Gating of Long-Range Radical Transport in Escherichia coli Class Ia Ribonucleotide Reductase.

Escherichia coli class Ia ribonucleotide reductase (RNR) is composed of two subunits that form an active α2ß2 complex. The nucleoside diphosphate substrates (NDP) are reduced in α2, 35 Å from the essential diferric-tyrosyl radical (Y122•) cofactor in ß2. The Y122•-mediated oxidation of C439 in α2 occurs by a pathway (Y122 ⇆ [W48] ⇆ Y356 in ß2 to Y731 ⇆ Y730 ⇆ C439 in α2) across the α/ß interface. The absence of an α2ß2 structure precludes insight into the location of Y356 and Y731 at the subunit interface. The proximity in the primary sequence of the conserved E350 to Y356 in ß2 suggested its importance in catalysis and/or conformational gating. To study its function, pH-rate profiles of wild-type ß2/α2 and mutants in which 3,5-difluorotyrosine (F2Y) replaces residue 356, 731, or both are reported in the presence of E350 or E350X (X = A, D, or Q) mutants. With E350, activity is maintained at the pH extremes, suggesting that protonated and deprotonated states of F2Y356 and F2Y731 are active and that radical transport (RT) can occur across the interface by proton-coupled electron transfer at low pH or electron transfer at high pH. With E350X mutants, all RNRs were inactive, suggesting that E350 could be a proton acceptor during oxidation of the interface Ys. To determine if E350 plays a role in conformational gating, the strong oxidants, NO2Y122•-ß2 and 2,3,5-F3Y122•-ß2, were reacted with α2, CDP, and ATP in E350 and E350X backgrounds and the reactions were monitored for pathway radicals by rapid freeze-quench electron paramagnetic resonance spectroscopy. Pathway radicals are generated only when E350 is present, supporting its essential role in gating the conformational change(s) that initiates RT and masking its role as a proton acceptor.

Q-Band Electron-Nuclear Double Resonance Reveals Out-of-Plane Hydrogen Bonds Stabilize an Anionic Ubisemiquinone in Cytochrome bo3 from Escherichia coli.

The respiratory cytochrome bo3 ubiquinol oxidase from Escherichia coli has a high-affinity ubiquinone binding site that stabilizes the one-electron reduced ubisemiquinone (SQH), which is a transient intermediate during the electron-mediated reduction of O2 to water. It is known that SQH is stabilized by two strong hydrogen bonds from R71 and D75 to ubiquinone carbonyl oxygen O1 and weak hydrogen bonds from H98 and Q101 to O4. In this work, SQH was investigated with orientation-selective Q-band (∼34 GHz) pulsed 1H electron-nuclear double resonance (ENDOR) spectroscopy on fully deuterated cytochrome (cyt) bo3 in a H2O solvent so that only exchangeable protons contribute to the observed ENDOR spectra. Simulations of the experimental ENDOR spectra provided the principal values and directions of the hyperfine (hfi) tensors for the two strongly coupled H-bond protons (H1 and H2). For H1, the largest principal component of the proton anisotropic hfi tensor Tz' = 11.8 MHz, whereas for H2, Tz' = 8.6 MHz. Remarkably, the data show that the direction of the H1 H-bond is nearly perpendicular to the quinone plane (∼70° out of plane). The orientation of the second strong hydrogen bond, H2, is out of plane by ∼25°. Equilibrium molecular dynamics simulations on a membrane-embedded model of the cyt bo3 QH site show that these H-bond orientations are plausible but do not distinguish which H-bond, from R71 or D75, is nearly perpendicular to the quinone ring. Density functional theory calculations support the idea that the distances and geometries of the H-bonds to the ubiquinone carbonyl oxygens, along with the measured proton anisotropic hfi couplings, are most compatible with an anionic (deprotonated) ubisemiquinone.

A >200 meV Uphill Thermodynamic Landscape for Radical Transport in Escherichia coli Ribonucleotide Reductase Determined Using Fluorotyrosine-Substituted Enzymes.

Escherichia coli class Ia ribonucleotide reductase (RNR) converts ribonucleotides to deoxynucleotides. A diferric-tyrosyl radical (Y122•) in one subunit (ß2) generates a transient thiyl radical in another subunit (α2) via long-range radical transport (RT) through aromatic amino acid residues (Y122 ⇆ [W48] ⇆ Y356 in ß2 to Y731 ⇆ Y730 ⇆ C439 in α2). Equilibration of Y356•, Y731•, and Y730• was recently observed using site specifically incorporated unnatural tyrosine analogs; however, equilibration between Y122• and Y356• has not been detected. Our recent report of Y356• formation in a kinetically and chemically competent fashion in the reaction of ß2 containing 2,3,5-trifluorotyrosine at Y122 (F3Y122•-ß2) with α2, CDP (substrate), and ATP (effector) has now afforded the opportunity to investigate equilibration of F3Y122• and Y356•. Incubation of F3Y122•-ß2, Y731F-α2 (or Y730F-α2), CDP, and ATP at different temperatures (2-37 °C) provides ΔE°'(F3Y122•-Y356•) of 20 ± 10 mV at 25 °C. The pH dependence of the F3Y122• ⇆ Y356• interconversion (pH 6.8-8.0) reveals that the proton from Y356 is in rapid exchange with solvent, in contrast to the proton from Y122. Insertion of 3,5-difluorotyrosine (F2Y) at Y356 and rapid freeze-quench EPR analysis of its reaction with Y731F-α2, CDP, and ATP at pH 8.2 and 25 °C shows F2Y356• generation by the native Y122•. FnY-RNRs (n = 2 and 3) together provide a model for the thermodynamic landscape of the RT pathway in which the reaction between Y122 and C439 is ∼200 meV uphill.