Ribonucleotide reductase (RNR) catalyzes the conversion of nucleoside diphosphates to deoxynucleoside diphosphates (dNDPs). The Escherichia coli class Ia RNR uses a mechanism of radical propagation by which a cysteine in the active site of the RNR large (α2) subunit is transiently oxidized by a stable tyrosyl radical (Y•) . These results present a structural and biochemical characterization of the active RNR complex "trapped" during turnover, and suggest that stabilization of the α2β2 state may be a regulatory mechanism for protecting the catalytic radical and ensuring the fidelity of its reactivity.conformational equilibria | radical transfer | unnatural amino acid R ibonucleotide reductase (RNR) is the sole enzyme responsible for the conversion of nucleoside diphosphates (NDPs) to 2′-deoxynucleoside diphosphates (dNDPs), providing the cell with the monomeric precursors necessary for DNA replication and repair (1, 2). The class I RNRs are composed of two subunits, α and β;the active form of the prototypical Escherichia coli class Ia enzyme is generally accepted to be α2β2 (3, 4). The α2 subunit houses the active site, where the four NDP substrates (CDP, ADP, GDP, and UDP) are reduced, and two distinct regulatory sites, where allosteric effectors (ATP, dGTP, dTTP, and dATP) bind. The specificity site dictates which of the four substrates is reduced, whereas the activity site binds ATP/dATP and regulates the overall rate of reduction (5). The β2 subunit, an obligate dimer, contains a diferric-tyrosyl radical cofactor (Y 122 •) that is essential for catalysis. Xray crystal structures of the individual E. coli β2 and α2 subunits have been solved (6, 7). However, the weak interaction between α2 and β2 (8), the conformational rearrangements induced by nucleotide binding (2, 9, 10), and complicated subunit equilibria (11) have precluded detailed structural characterization of any active RNR complexes. We now report the characterization of an active, kinetically stable α2β2 complex that forms transiently during turnover.Nearly 2 decades ago, Uhlin and Eklund (7) put forth a docking model for the active E. coli α2β2 complex based on shape complementarity between the structures of the individual subunits.Their model predicted a 35-Å distance between the diferric-Y 122 • cofactor in β2 and the active site cysteine (C 439 ) in α2, the transient oxidation of which is a prerequisite for nucleotide reduction (1). A radical transfer (RT) pathway of conserved aromatic amino acids was proposed to account for kinetically competent radical propagation over this long distance (7). The thermodynamics of Y oxidation require loss of a proton to accompany loss of an electron, and the more detailed mechanism for proton-coupled electron transfer shown in Fig. 1A has emerged from experiments conducted in our laboratories (12,13).Evidence for the utilization of an amino acid pathway in longrange RT has been derived from several types of experiments. Initial site-directed mutagenesis studies of the conserved residues (Fig. 1A) supported t...

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.

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.

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.

The interplay between oxidation state and coordination geometry dictates both kinetic and thermodynamic properties underlying electron transfer events in copper complexes. An ability to stabilize both CuI and CuII oxidation...

Artificial metalloproteins (ArMs) containing Co4O4 cubane active sites were constructed via biotin-streptavidin technology. Stabilized by hydrogen bonds (H-bonds), terminal and cofacial CoIII–OH2 moieties are observed crystallographically in a series of immobilized cubane sites. Solution electrochemistry provided correlations of oxidation potential and pH. For variants containing Ser and Phe adjacent to the metallocofactor, 1e−/1H+ chemistry predominates until pH 8, above which the oxidation becomes pH-independent. Installation of Tyr proximal to the Co4O4 active site provided a single H-bond to one of a set of cofacial CoIII–OH2 groups. With this variant, multi-e−/multi-H+ chemistry is observed, along with a change in mechanism at pH 9.5 that is consistent with Tyr deprotonation. With structural similarities to both the oxygen-evolving complex of photosystem II (H-bonded Tyr) and to amorphous water oxidation catalysts (Co4O4 core), these findings bridge synthetic and biological systems for water oxidation, highlighting the importance of secondary sphere interactions in mediating multi-e−/multi-H+ reactivity.

The continued development of solar energy as a renewable resource necessitates new approaches to sustaining photodriven charge separation (CS). We present a bioinspired approach in which photoinduced conformational rearrangements at a ligand are translated into changes in coordination geometry and environment about a bound metal ion. Taking advantage of the differential coordination properties of CuI and CuII, these dynamics aim to facilitate intramolecular electron transfer (ET) from CuI to the ligand to create a CS state. The synthesis and photophysical characterization of CuCl­(dpaaR) (dpaa = dipicolylaminoacetophenone, with R = H and OMe) are presented. These ligands incorporate a fluorophore that gives rise to a twisted intramolecular charge transfer (TICT) excited state. Excited-state ligand twisting provides a tetragonal coordination geometry capable of capturing CuII when an internal ortho-OMe binding site is present. NMR, IR, electron paramagnetic resonance (EPR), and optical spectroscopies, X-ray diffraction, and electrochemical methods establish the ground-state properties of these CuI and CuII complexes. The photophysical dynamics of the CuI complexes are explored by time-resolved photoluminescence and optical transient absorption spectroscopies. Relative to control complexes lacking a TICT-active ligand, the lifetimes of CS states are enhanced ∼1000-fold. Further, the presence of the ortho-OMe substituent greatly enhances the lifetime of the TICT* state and biases the coordination environment toward CuII. The presence of CuI decreases photoinduced degradation from 14 to <2% but does not result in significant quenching via ET. Factors affecting CS in these systems are discussed, laying the groundwork for our strategy toward solar energy conversion.

Active site hydrogen-bond (H-bond) networks represent a key component by which metalloenzymes control the formation and deployment of high-valent transition metal-oxo intermediates. We report a series of dinuclear cobalt complexes that serve as structural models for the nonheme diiron enzyme family and feature a Co 2 (μ−OH) 2 diamond core stabilized by intramolecular H-bond interactions. We define the conditions required for the kinetically controlled synthesis of these complexes:where OAc = acetate and py R = pyridine with para-substituent R, and we describe a homologous series of 1 R in which the para-R substituent on pyridine is modulated. The solid state X-ray diffraction (XRD) structures of 1 R are similar across the series, but in solution, their 1 H NMR spectra reveal a linear free energy relationship (LFER) where, as R becomes increasingly electron-withdrawing, the intramolecular H-bond interaction between bridging μ−OH and κ 1 -acetate ligands results in increasingly "oxo-like" μ−OH bridges. Deprotonation of the bridging μ− OH results in the quantitative conversion to corresponding cubane complexes: [Co 4 (μ-O) 4 (μ 3 -OAc) 4 (py R ) 4 ] (2 R ), which represent the thermodynamic sink of self-assembly. These reactions are unusually slow for rate-limiting deprotonation events, but rapid-mixing experiments reveal a 6000-fold rate acceleration on going from R = OMe to R = CN. These results suggest that we can tune reactivity by modulating the μ−OH pK a in the presence of intramolecular H-bond interactions to maintain stability as the octahedral d 6 centers become increasingly acidic. Nature may similarly employ dynamic carboxylate-mediated H-bond interactions to control the reactivity of acidic transition metal-oxo intermediates.