Long-range Proton-coupled Electron Transfer Research Articles

Radical Transport Facilitated by a Proton Transfer Network at the Subunit Interface of Ribonucleotide Reductase.

Ribonucleotide reductases (RNRs) play an essential role in the conversion of nucleotides to deoxynucleotides in all organisms. The Escherichia coli class Ia RNR requires two homodimeric subunits, α and β. The active form is an asymmetric αα'ββ' complex. The α subunit houses the site for nucleotide reduction initiated by a thiyl radical (C439•), and the β subunit houses the diferric-tyrosyl radical (Y122•) that is essential for C439• formation. The reactions require a highly regulated and reversible long-range proton-coupled electron transfer pathway involving Y122•[β] ↔ W48?[β] ↔ Y356[β] ↔ Y731[α] ↔ Y730[α] ↔ C439[α]. In a recent cryo-EM structure, Y356[β] was revealed for the first time and it, along with Y731[α], spans the asymmetric α/β interface. An E52[β] residue, which is essential for Y356 oxidation, allows access to the interface and resides at the head of a polar region comprising R331[α], E326[α], and E326[α'] residues. Mutagenesis studies with canonical and unnatural amino acid substitutions now suggest that these ionizable residues are important in enzyme activity. To gain further insights into the roles of these residues, Y356• was photochemically generated using a photosensitizer covalently attached adjacent to Y356[β]. Mutagenesis studies, transient absorption spectroscopy, and photochemical assays monitoring deoxynucleotide formation collectively indicate that the E52[β], R331[α], E326[α], and E326[α'] network plays the essential role of shuttling protons associated with Y356 oxidation from the interface to bulk solvent.

Resolving Chemical Dynamics in Biological Energy Conversion: Long-Range Proton-Coupled Electron Transfer in Respiratory Complex I.

ConspectusBiological energy conversion is catalyzed by membrane-bound proteins that transduce chemical or light energy into energy forms that power endergonic processes in the cell. At a molecular level, these catalytic processes involve elementary electron-, proton-, charge-, and energy-transfer reactions that take place in the intricate molecular machineries of cell respiration and photosynthesis. Recent developments in structural biology, particularly cryo-electron microscopy (cryoEM), have resolved the molecular architecture of several energy transducing proteins, but detailed mechanistic principles of their charge transfer reactions still remain poorly understood and a major challenge for modern biochemical research. To this end, multiscale molecular simulations provide a powerful approach to probe mechanistic principles on a broad range of time scales (femtoseconds to milliseconds) and spatial resolutions (101–106 atoms), although technical challenges also require balancing between the computational accuracy, cost, and approximations introduced within the model. Here we discuss how the combination of atomistic (aMD) and hybrid quantum/classical molecular dynamics (QM/MM MD) simulations with free energy (FE) sampling methods can be used to probe mechanistic principles of enzymes responsible for biological energy conversion. We present mechanistic explorations of long-range proton-coupled electron transfer (PCET) dynamics in the highly intricate respiratory chain enzyme Complex I, which functions as a redox-driven proton pump in bacterial and mitochondrial respiratory chains by catalyzing a 300 Å fully reversible PCET process. This process is initiated by a hydride (H–) transfer between NADH and FMN, followed by long-range (>100 Å) electron transfer along a wire of 8 FeS centers leading to a quinone biding site. The reduction of the quinone to quinol initiates dissociation of the latter to a second membrane-bound binding site, and triggers proton pumping across the membrane domain of complex I, in subunits up to 200 Å away from the active site. Our simulations across different size and time scales suggest that transient charge transfer reactions lead to changes in the internal hydration state of key regions, local electric fields, and the conformation of conserved ion pairs, which in turn modulate the dynamics of functional steps along the reaction cycle. Similar functional principles, which operate on much shorter length scales, are also found in some unrelated proteins, suggesting that enzymes may employ conserved principles in the catalysis of biological energy transduction processes.

Detection of Catalytically Linked Conformational Changes in Wild-Type Class Ia Ribonucleotide Reductase Using Reaction-Induced FTIR Spectroscopy

The enzyme, ribonucleotide reductase (RNR), is essential for DNA synthesis in all cells. The class Ia Escherichia coli RNR consists of two dimeric subunits, α2 and β2, which form an active but unstable heterodimer of dimers, α2β2. The structure of the wild-type form of the enzyme has been challenging to study due to the instability of the catalytic complex. A long-range proton-coupled electron-transfer (PCET) pathway facilitates radical migration from the Y122 radical-diiron cofactor in the β subunit to an active site cysteine, C439, in the α subunit to initiate the RNR chemistry. The PCET reactions and active site chemistry are spectroscopically masked by a rate-limiting, conformational gate. Here, we present a reaction-induced Fourier transform infrared (RIFTIR) spectroscopic method to monitor the mechanism of the active, wild-type RNR α2β2 complex. This method is employed to obtain new information about conformational changes accompanying RNR catalysis, including the role of carboxylate interactions, deprotonation, and oxidation of active site cysteines, and a detailed description of reversible secondary structural changes. Labeling of tyrosine revealed a conformationally active tyrosine in the β subunit, assigned to Y356β, which is part of the intersubunit PCET pathway. New insights into the roles of the inhibitors, azidoUDP and dATP, and the sensitivity of RIFTIR spectroscopy to detect subtle conformational motions arising from protein allostery are also presented.

Functional Water Wires Catalyze Long-Range Proton Pumping in the Mammalian Respiratory Complex I.

The respiratory complex I is a gigantic (1 MDa) redox-driven proton pump that reduces the ubiquinone pool and generates proton motive force to power ATP synthesis in mitochondria. Despite resolved molecular structures and biochemical characterization of the enzyme from multiple organisms, its long-range (∼300 Å) proton-coupled electron transfer (PCET) mechanism remains unsolved. We employ here microsecond molecular dynamics simulations to probe the dynamics of the mammalian complex I in combination with hybrid quantum/classical (QM/MM) free energy calculations to explore how proton pumping reactions are triggered within its 200 Å wide membrane domain. Our simulations predict extensive hydration dynamics of the antiporter-like subunits in complex I that enable lateral proton transfer reactions on a microsecond time scale. We further show how the coupling between conserved ion pairs and charged residues modulate the proton transfer dynamics, and how transmembrane helices and gating residues control the hydration process. Our findings suggest that the mammalian complex I pumps protons by tightly linked conformational and electrostatic coupling principles.

The roles of long-range proton-coupled electron transfer in the directionality and efficiency of [FeFe]-hydrogenases

As paradigms for proton-coupled electron transfer in enzymes and benchmarks for a fully renewable H2 technology, [FeFe]-hydrogenases behave as highly reversible electrocatalysts when immobilized on an electrode, operating in both catalytic directions with minimal overpotential requirement. Using the [FeFe]-hydrogenases from Clostridium pasteurianum (CpI) and Chlamydomonas reinhardtii (CrHydA1) we have conducted site-directed mutagenesis and protein film electrochemistry to determine how efficient catalysis depends on the long-range coupling of electron and proton transfer steps. Importantly, the electron and proton transfer pathways in [FeFe]-hydrogenases are well separated from each other in space. Variants with conservative substitutions (glutamate to aspartate) in either of two positions in the proton-transfer pathway retain significant activity and reveal the consequences of slowing down proton transfer for both catalytic directions over a wide range of pH and potential values. Proton reduction in the variants is impaired mainly by limiting the turnover rate, which drops sharply as the pH is raised, showing that proton capture from bulk solvent becomes critical. In contrast, hydrogen oxidation is affected in two ways: by limiting the turnover rate and by a large overpotential requirement that increases as the pH is raised, consistent with the accumulation of a reduced and protonated intermediate. A unique observation having fundamental significance is made under conditions where the variants still retain sufficient catalytic activity in both directions: An inflection appears as the catalytic current switches direction at the 2H+/H2 thermodynamic potential, clearly signaling a departure from electrocatalytic reversibility as electron and proton transfers begin to be decoupled.

Ultrasmall Silver Clusters Stabilized on MgO for Robust Oxygen-Promoted Hydrogen Production from Formaldehyde Reforming.

Efficient molecular hydrogen generation from renewable biomass-derived resources and water is of great importance to the sustainable development of the future society. Herein, ultrasmall Ag nanoclusters supported on a defect-rich MgO matrix (AgUCs/MgO) are synthesized by a facile impregnation/calcination method and are applied to robust oxygen-promoted formaldehyde reforming into H2 at room temperature. Density functional theory calculations and experimental observations show that the catalyst spatially builds up a channel for directional electron transfer from electron-rich Ag sites to the anti-bonding π orbital of chemisorbed bridged O2 molecules, leading to the implementation of low-temperature O2 adsorption and activation. The catalytically active species, •OOH, is thus selectively generated via a preferential two-electron reduction of O2 with a low energy barrier on Ag sites, involving an unusual long-range proton-coupled electron transfer process. The •OOH-AgUCs/MgO active center is efficient for the subsequent C-H activation and H2 generation, leading to a 3-fold improvement of the turnover frequency as compared with its analogous AgNPs/MgO catalyst. Our atomic-level design and synthetic strategy provide a platform that facilitates the construction of an electron-proton transfer channel for catalysis, altered adsorption configurations of activated reactants, and enhancement of catalytic hydrogen generation activity, extending a promising direction for the development of next-generation energy catalysts.

Mechanistic Exploitation of a Self-Repairing, Blocked Proton Transfer Pathway in an O2-Tolerant [NiFe]-Hydrogenase.

Catalytic long-range proton transfer in [NiFe]-hydrogenases has long been associated with a highly conserved glutamate (E) situated within 4 Å of the active site. Substituting for glutamine (Q) in the O2-tolerant [NiFe]-hydrogenase-1 from Escherichia coli produces a variant (E28Q) with unique properties that have been investigated using protein film electrochemistry, protein film infrared electrochemistry, and X-ray crystallography. At pH 7 and moderate potential, E28Q displays approximately 1% of the activity of the native enzyme, high enough to allow detailed infrared measurements under steady-state conditions. Atomic-level crystal structures reveal partial displacement of the amide side chain by a hydroxide ion, the occupancy of which increases with pH or under oxidizing conditions supporting formation of the superoxidized state of the unusual proximal [4Fe-3S] cluster located nearby. Under these special conditions, the essential exit pathway for at least one of the H+ ions produced by H2 oxidation, and assumed to be blocked in the E28Q variant, is partially repaired. During steady-state H2 oxidation at neutral pH (i.e., when the barrier to H+ exit via Q28 is almost totally closed), the catalytic cycle is dominated by the reduced states "Nia-R" and "Nia-C", even under highly oxidizing conditions. Hence, E28 is not involved in the initial activation/deprotonation of H2, but facilitates H+ exit later in the catalytic cycle to regenerate the initial oxidized active state, assumed to be Nia-SI. Accordingly, the oxidized inactive resting state, "Ni-B", is not produced by E28Q in the presence of H2 at high potential because Nia-SI (the precursor for Ni-B) cannot accumulate. The results have important implications for understanding the catalytic mechanism of [NiFe]-hydrogenases and the control of long-range proton-coupled electron transfer in hydrogenases and other enzymes.

Long-range proton-coupled electron transfer in biological energy conversion: towards mechanistic understanding of respiratory complex I.

Biological energy conversion is driven by efficient enzymes that capture, store and transfer protons and electrons across large distances. Recent advances in structural biology have provided atomic-scale blueprints of these types of remarkable molecular machinery, which together with biochemical, biophysical and computational experiments allow us to derive detailed energy transduction mechanisms for the first time. Here, I present one of the most intricate and least understood types of biological energy conversion machinery, the respiratory complex I, and how its redox-driven proton-pump catalyses charge transfer across approximately 300 Å distances. After discussing the functional elements of complex I, a putative mechanistic model for its action-at-a-distance effect is presented, and functional parallels are drawn to other redox- and light-driven ion pumps.

Symmetry-related proton transfer pathways in respiratory complex I

Complex I functions as the initial electron acceptor in aerobic respiratory chains of most organisms. This gigantic redox-driven enzyme employs the energy from quinone reduction to pump protons across its complete approximately 200-Å membrane domain, thermodynamically driving synthesis of ATP. Despite recently resolved structures from several species, the molecular mechanism by which complex I catalyzes this long-range proton-coupled electron transfer process, however, still remains unclear. We perform here large-scale classical and quantum molecular simulations to study the function of the proton pump in complex I from Thermus thermophilus The simulations suggest that proton channels are established at symmetry-related locations in four subunits of the membrane domain. The channels open up by formation of quasi one-dimensional water chains that are sensitive to the protonation states of buried residues at structurally conserved broken helix elements. Our combined data provide mechanistic insight into long-range coupling effects and predictions for site-directed mutagenesis experiments.

Kinetics of hydrogen atom abstraction from substrate by an active site thiyl radical in ribonucleotide reductase.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. Active E. coli class Ia RNR is an α2β2 complex that undergoes reversible, long-range proton-coupled electron transfer (PCET) over a pathway of redox active amino acids (β-Y122 → [β-W48] → β-Y356 → α-Y731 → α-Y730 → α-C439) that spans ∼35 Å. To unmask PCET kinetics from rate-limiting conformational changes, we prepared a photochemical RNR containing a [ReI] photooxidant site-specifically incorporated at position 355 ([Re]-β2), adjacent to PCET pathway residue Y356 in β. [Re]-β2 was further modified by replacing Y356 with 2,3,5-trifluorotyrosine to enable photochemical generation and spectroscopic observation of chemically competent tyrosyl radical(s). Using transient absorption spectroscopy, we compare the kinetics of Y· decay in the presence of substrate and wt-α2, Y731F-α2 ,or C439S-α2, as well as with 3′-[2H]-substrate and wt-α2. We find that only in the presence of wt-α2 and the unlabeled substrate do we observe an enhanced rate of radical decay indicative of forward radical propagation. This observation reveals that cleavage of the 3′-C–H bond of substrate by the transiently formed C439· thiyl radical is rate-limiting in forward PCET through α and has allowed calculation of a lower bound for the rate constant associated with this step of (1.4 ± 0.4) × 104 s–1. Prompting radical propagation with light has enabled observation of PCET events heretofore inaccessible, revealing active site chemistry at the heart of RNR catalysis.

Tyrosine oxidation in heme oxygenase: examination of long-range proton-coupled electron transfer.

Heme oxygenase is responsible for the degradation of a histidine-ligated ferric protoporphyrin IX (Por) to biliverdin, CO, and the free ferrous ion. Described here are studies of tyrosyl radical formation reactions that occur after oxidizing Fe(III)(Por) to Fe(IV)=O(Por(·+)) in human heme oxygenase isoform-1 (hHO-1) and the structurally homologous protein from Corynebacterium diphtheriae (cdHO). Site-directed mutagenesis on hHO-1 probes the reduction of Fe(IV)=O(Por(·+)) by tyrosine residues within 11 Å of the prosthetic group. In hHO-1, Y58· is implicated as the most likely site of oxidation, based on the pH and pD dependent kinetics. The absence of solvent deuterium isotope effects in basic solutions of hHO-1 and cdHO contrasts with the behavior of these proteins in the acidic solution, suggesting that long-range proton-coupled electron transfer predominates over electron transfer.

Electrostatics, hydration, and proton transfer dynamics in the membrane domain of respiratory complex I.

Complex I serves as the primary electron entry point into the mitochondrial and bacterial respiratory chains. It catalyzes the reduction of quinones by electron transfer from NADH, and couples this exergonic reaction to the translocation of protons against an electrochemical proton gradient. The membrane domain of the enzyme extends ∼180 Å from the site of quinone reduction to the most distant proton pathway. To elucidate possible mechanisms of the long-range proton-coupled electron transfer process, we perform large-scale atomistic molecular dynamics simulations of the membrane domain of complex I from Escherichia coli. We observe spontaneous hydration of a putative proton entry channel at the NuoN/K interface, which is sensitive to the protonation state of buried glutamic acid residues. In hybrid quantum mechanics/classical mechanics simulations, we find that the observed water wires support rapid proton transfer from the protein surface to the center of the membrane domain. To explore the functional relevance of the pseudosymmetric inverted-repeat structures of the antiporter-like subunits NuoL/M/N, we constructed a symmetry-related structure of a possible alternate-access state. In molecular dynamics simulations, we find the resulting structural changes to be metastable and reversible at the protein backbone level. However, the increased hydration induced by the conformational change persists, with water molecules establishing enhanced lateral connectivity and pathways for proton transfer between conserved ionizable residues along the center of the membrane domain. Overall, the observed water-gated transitions establish conduits for the unidirectional proton translocation processes, and provide a possible coupling mechanism for the energy transduction in complex I.

Long-range proton-coupled electron transfer in phenol–Ru(2,2′-bipyrazine)32+ dyads

Two dyads in which either 4-cyanophenol or un-substituted phenol is connected via a p-xylene spacer to a Ru(bpz)3(2+) (bpz = 2,2'-bipyrazine) complex were synthesized and investigated. Selective photo-excitation of Ru(bpz)3(2+) at 532 nm in a CH3CN-H2O mixture leads to the formation of 4-cyanophenolate or phenolate along with Ru(bpz)3(2+) in its electronic ground state. This apparent photoacid behavior can be understood on the basis of a reaction sequence comprised of an initial photoinduced proton-coupled electron transfer (PCET) during which 4-cyanophenol or phenol is oxidized and deprotonated, followed by a thermal electron transfer event in the course of which 4-cyanophenoxyl or phenoxyl is reduced by Ru(bpz)3(+) to 4-cyanophenolate or phenolate. Conceptually, this reaction sequence is identical to a sequence of photoinduced charge-separation and thermal charge-recombination events as observed previously for many electron transfer dyads, with the important difference that the initial photoinduced electron transfer process is proton-coupled. The dyad containing 4-cyanophenol reacts via concerted-proton electron transfer (CPET) whereas the dyad containing un-substituted phenol appears to react predominantly via a stepwise PCET mechanism. Long-range PCET is a key reaction in photosystem II. Understanding the factors that govern the kinetics of long-range PCET is desirable in the broader context of light-to-energy conversion by means of proton-electron separation across natural or artificial membranes.

Water Gated Transitions in Proton Pumping of Respiratory Complex I

Respiratory Complex I or NADH:ubiquinone oxidoreductase is a redox-driven proton-pump. Powered by quinone reduction, Complex I drives the energetically uphill translocation of protons across the mitochondrial inner membrane and bacterial cytoplasmic membrane. The established electrochemical proton gradient provides the driving force for active transport and synthesis of ATP and is thus crucial for biological energy conversion. Complex I comprises a membrane domain that includes three antiporter-like subunits, involved in the proton-pumping process, and a soluble domain, responsible for reduction of quinones by electron transfer from NADH. Remarkably, site-directed mutagenesis experiments show that mutations of titratable residues in the antiporter-like subunits, ∼200 A away from site of quinone reduction, inhibit both proton pumping and quinone reduction. To explain this long-range proton-coupled electron transfer mechanism, both indirect and direct coupling models have been suggested. However, despite the recent elucidation of the complete intact structure of Complex I, the molecular principles of the coupling principles remain elusive. We present here results of large-scale classical and hybrid quantum-classical (QM/MM) molecular dynamics (MD) simulations of Complex I, embedded in biologically realistic environments. Our simulations indicate that water molecules provide important elements in the proton-pumping process. Our findings may form a basis for understanding long-range energy transduction in Complex I, and mechanistic similarities to other redox-driven proton-pumps such as cytochrome c oxidase and bacteriorhodopsin.

The Role of Water‐Based Hydrogen Atom Wires in Long‐Range Electron‐Transfer Reactions in Aqueous Media for the FeII–FeIII Self‐Exchange and Related Systems

Extended reach: A calculated mechanism for long-range proton-coupled electron transfer (PCET, see picture) through an array of structured water molecules between Fe(III)-Fe(III) complexes accounts for the reaction enthalpy and kinetic isotope effect previously measured for this reaction. This mechanism may be general and occur for other hydroxo-metal complexes.

Equilibration of tyrosyl radicals (Y356•, Y731•, Y730•) in the radical propagation pathway of the Escherichia coli class Ia ribonucleotide reductase.

Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides using a diferric tyrosyl radical (Y(122)(•)) cofactor in β2 to initiate catalysis in α2. Each turnover requires reversible long-range proton-coupled electron transfer (PCET) over 35 Å between the two subunits by a specific pathway (Y(122)(•) ⇆ [W(48)?] ⇆ Y(356) within β to Y(731) ⇆ Y(730) ⇆ C(439) within α). Previously, we reported that a β2 mutant with 3-nitrotyrosyl radical (NO(2)Y(•); 1.2 radicals/β2) in place of Y(122)(•) in the presence of α2, CDP, and ATP catalyzes formation of 0.6 equiv of dCDP and accumulates 0.6 equiv of a new Y(•) proposed to be located on Y(356) in β2. We now report three independent methods that establish that Y(356) is the predominant location (85-90%) of the radical, with the remaining 10-15% delocalized onto Y(731) and Y(730) in α2. Pulsed electron-electron double-resonance spectroscopy on samples prepared by rapid freeze quench (RFQ) methods identified three distances: 30 ± 0.4 Å (88% ± 3%) and 33 ± 0.4 and 38 ± 0.5 Å (12% ± 3%) indicative of NO(2)Y(122)(•)-Y(356)(•), NO(2)Y(122)(•)-NO(2)Y(122)(•), and NO(2)Y(122)(•)-Y(731(730))(•), respectively. Radical distribution in α2 was supported by RFQ electron paramagnetic resonance (EPR) studies using Y(731)(3,5-F(2)Y) or Y(730)(3,5-F(2)Y)-α2, which revealed F(2)Y(•), studies using globally incorporated [β-(2)H(2)]Y-α2, and analysis using parameters obtained from 140 GHz EPR spectroscopy. The amount of Y(•) delocalized in α2 from these two studies varied from 6% to 15%. The studies together give the first insight into the relative redox potentials of the three transient Y(•) radicals in the PCET pathway and their conformations.

Water‐Based Hydrogen‐Atom Wires as Mediators in Long‐Range Proton‐Coupled Electron Transfer in Enzymes: A New Twist on Water Reactivity

Water‐Based Hydrogen‐Atom Wires as Mediators in Long‐Range Proton‐Coupled Electron Transfer in Enzymes: A New Twist on Water Reactivity

Kinetics of radical intermediate formation and deoxynucleotide production in 3-aminotyrosine-substituted Escherichia coli ribonucleotide reductases.

Escherichia coli ribonucleotide reductase is an α2β2 complex and catalyzes the conversion of nucleoside 5'-diphosphates (NDPs) to 2'-deoxynucleotides (dNDPs). The reaction is initiated by the transient oxidation of an active-site cysteine (C(439)) in α2 by a stable diferric tyrosyl radical (Y(122)•) cofactor in β2. This oxidation occurs by a mechanism of long-range proton-coupled electron transfer (PCET) over 35 Å through a specific pathway of residues: Y(122)•→ W(48)→ Y(356) in β2 to Y(731)→ Y(730)→ C(439) in α2. To study the details of this process, 3-aminotyrosine (NH(2)Y) has been site-specifically incorporated in place of Y(356) of β. The resulting protein, Y(356)NH(2)Y-β2, and the previously generated proteins Y(731)NH(2)Y-α2 and Y(730)NH(2)Y-α2 (NH(2)Y-RNRs) are shown to catalyze dNDP production in the presence of the second subunit, substrate (S), and allosteric effector (E) with turnover numbers of 0.2-0.7 s(-1). Evidence acquired by three different methods indicates that the catalytic activity is inherent to NH(2)Y-RNRs and not the result of copurifying wt enzyme. The kinetics of formation of 3-aminotyrosyl radical (NH(2)Y•) at position 356, 731, and 730 have been measured with all S/E pairs. In all cases, NH(2)Y• formation is biphasic (k(fast) of 9-46 s(-1) and k(slow) of 1.5-5.0 s(-1)) and kinetically competent to be an intermediate in nucleotide reduction. The slow phase is proposed to report on the conformational gating of NH(2)Y• formation, while the k(cat) of ~0.5 s(-1) is proposed to be associated with rate-limiting oxidation by NH(2)Y• of the subsequent amino acid on the pathway during forward PCET. The X-ray crystal structures of Y(730)NH(2)Y-α2 and Y(731)NH(2)Y-α2 have been solved and indicate minimal structural changes relative to wt-α2. From the data, a kinetic model for PCET along the radical propagation pathway is proposed.

A hot oxidant, 3-NO2Y122 radical, unmasks conformational gating in ribonucleotide reductase.

Escherichia coli ribonucleotide reductase is an α2β2 complex that catalyzes the conversion of nucleotides to deoxynucleotides and requires a diferric-tyrosyl radical (Y(•)) cofactor to initiate catalysis. The initiation process requires long-range proton-coupled electron transfer (PCET) over 35 Å between the two subunits by a specific pathway (Y(122)(•)→W(48)→Y(356) within β to Y(731)→Y(730)→C(439) within α). The rate-limiting step in nucleotide reduction is the conformational gating of the PCET process, which masks the chemistry of radical propagation. 3-Nitrotyrosine (NO(2)Y) has recently been incorporated site-specifically in place of Y(122) in β2. The protein as isolated contained a diferric cluster but no nitrotyrosyl radical (NO(2)Y(•)) and was inactive. In the present paper we show that incubation of apo-Y(122)NO(2)Y-β2 with Fe(2+) and O(2) generates a diferric-NO(2)Y(•) that has a half-life of 40 s at 25 °C. Sequential mixing experiments, in which the cofactor is assembled to 1.2 NO(2)Y(•)/β2 and then mixed with α2, CDP, and ATP, have been analyzed by stopped-flow absorption spectroscopy, rapid freeze quench EPR spectroscopy, and rapid chemical quench methods. These studies have, for the first time, unmasked the conformational gating. They reveal that the NO(2)Y(•) is reduced to the nitrotyrosinate with biphasic kinetics (283 and 67 s(-1)), that dCDP is produced at 107 s(-1), and that a new Y(•) is produced at 97 s(-1). Studies with pathway mutants suggest that the new Y(•) is predominantly located at 356 in β2. In consideration of these data and the crystal structure of Y(122)NO(2)Y-β2, a mechanism for PCET uncoupling in NO(2)Y(•)-RNR is proposed.

Tyrosine Analogues for Probing Proton-Coupled Electron Transfer Processes in Peptides and Proteins

A series of amino acids analogous to tyrosine, but differing in the physicochemical properties of the aryl alcohol side chain, have been prepared and characterized. These compounds are expected to be useful in understanding the relationships between structure, thermodynamics, and kinetics in long-range proton-coupled electron transfer processes in peptides and proteins. Systematic changes in the acidity, redox potential, and O-H bond strength of the tyrosine side chain could be induced upon substituting the phenol for pyridinol and pyrimidinol moieties. Further modulation was possible by introducing methyl and t-butyl substitution in the position ortho to the phenolic hydroxyl. The unnatural amino acids were prepared by Pd-catalyzed cross-coupling of the corresponding halogenated aryl alcohol protected as their benzyl ethers with an organozinc reagent derived from N-Boc L-serine carboxymethyl ester. Subsequent debenzylation by catalytic hydrogenation yielded the tyrosine analogues in good yield. Spectrophotometric titrations revealed a decrease in tyrosine pK(a) of ca. 1.5 log units per included nitrogen atom, along with a corresponding increase in the oxidation (peak) potentials of ca. 200 mV, respectively. All told, the six novel amino acids described here have phenol-like side chains with pK(a)'s that span a range of 7.0 to greater than 10, and an oxidation (peak) potential range of greater than 600 mV at and around physiological pH. Radical equilibration EPR experiments were carried out to reveal that the O-H bond strengths increase systematically upon nitrogen incorporation (by ca. 0.5-1.0 kcal/mol), and radical stability and persistence increase systematically upon introduction of alkyl substitution in the ortho positions. The EPR spectra of the aryloxyl radicals derived from tyrosine and each of the analogues could be determined at room temperature, and each featured distinct spectral properties. The uniqueness of their spectra will be helpful in discerning one type of aryloxyl in the presence of other possible aryloxyl radicals in peptides and proteins with multiple tyrosine residues between which electrons and protons can be transferred.