Carboxylate Shift Dynamics in Biomimetic Co2(µ-OH)2 Complexes.

Carboxylate shift mechanisms provide low-energy pathways to accommodate changes in oxidation state and coordination number required during catalysis in metalloenzyme active sites. These processes are challenging to observe in their native enzymes and molecular models can provide insight into their mechanistic details. We report here the direct observation of a carboxylate shift reaction in biomimetic yet structurally stable dicobalt complexes featuring both monodentate and bridging acetate ligands, as well as intramolecular hydrogen-bonding interactions. Subjecting the series of complexes [Co2(µ-OH)2(µ-1,3-OAc)(κ-OAc)2(pyR)4]PF6 ([1R]PF6, OAc = acetate, pyR = pyridine with para-R substituents: OMe, H, or CN) to a Lewis acid triggers conversion of a monodentate acetate to a µ-1,3 bridging mode, forming [Co2(µ-OH)2(µ-1,3-OAc)2(pyR)4]2+ ([2R]2+). [2R]2+ is susceptible to solvent binding, affording [Co2(µ-OH)2(µ-1,3-OAc)(κ-OAc)(MeCN)(pyR)4]2+ ([3R]2+) in MeCN. These reaction products and intermediates were isolated and characterized in the solid state by isotopic labeling and Fourier transform infrared (FTIR) spectroscopy, as well as by X-ray diffraction. The kinetics of the formation and decay of [1R]+, [2R]2+, and [3R]2+ were also examined in situ by 1H-NMR spectroscopy to provide a kinetic model for the carboxylate shift reaction. The rate constants extracted from global fit analyses of these reactions increase with increasing electron donation from R. Leveraging robust diamagnetic CoIII complexes, these studies provide mechanistic details of carboxylate shift reactivity and highlight the utility of ligand dynamicity in mediating the transient formation of unstable metal complexes.

Excited State Dynamics of a Conformationally Fluxional Copper Coordination Complex.

The conversion of solar energy into chemical fuel represents a capstone goal of the 21st century and has the potential to supply terawatts of power in a globally distributed manner. However, the disparate time scales of photodriven charge separation (∼fs) and steps in chemical reactions (∼µs) represent an inherent bottleneck in solar-to-fuels technology. To address this discrepancy, we are developing earth-abundant coordination complexes that undergo light-induced conformational rearrangements such that charge separation (CS) is hastened, while charge recombination (CR) is slowed. To these ends, we report the preparation and characterization of a new series of conformationally fluxional copper coordination complexes that contain a twisted intramolecular charge transfer (TICT) fluorophore as part of their ligand scaffold. Structural and spectroscopic characterization of the Cu(I) and Cu(II) complexes formed with these ligands in their ground states establish oxidation state-dependent conformational dynamicity, while time-resolved emission and transient absorption spectroscopies define the photophysical parameters of photo-induced excited states. Building on initial reports with a related set of molecules, the improved ligand design presented here greatly simplifies the observed photophysics, effectively shutting down unwanted ligand-centered excited states previously observed. Time-dependent density functional theory (TDDFT) analyses reveal an unusual metal-to-TICT electronic transition only reported once before, and though the formation of a CS state is not observed directly through experiments, TDDFT geometry optimizations in the excited states support the formation of transient Cu(II) CS species, lending credence to the potential success of our approach. These studies establish a clear model for the excited state dynamics at play in proof-of-concept systems and clarify key design parameters for future optimizations toward achieving long-lived CS via photoinduced conformational gating.

Rapid Electron Transfer Self-Exchange in Conformationally Dynamic Copper Coordination Complexes.

We report the electron transfer (ET) self-exchange rate constants (k11) for a pair of CuII/I complexes utilizing dpaR (dpa = dipicolylaniline, R = OMe, SMe) ligands assessed by NMR line broadening experiments. These ligands afford copper complexes that are conformationally dynamic in one oxidation state. With R = OMe, the CuI complex is dynamic, while with R = SMe, the CuII complex is dynamic. Both complexes exhibit unexpectedly large k11 values of 2.48(6) × 105 and 2.21(9) × 106 M-1 s-1 for [CuCl(dpaOMe)]+/0 and [CuCl(dpaSMe)]+/0, respectively. Among the fastest reported molecular copper coordination complexes to date, that of [CuCl(dpaSMe)]+/0 exceeds all others by an order of magnitude and compares only with those observed in type 1 blue copper proteins. The dynamicity of these complexes establishes pre-steady-state conformational equilibria that minimize the inner-sphere reorganization energies to 0.71 and 0.62 eV for R = OMe and SMe, respectively. In contrast to the emphasis on rigidity in the formulation of entatic states applied to blue copper proteins, the success of these two systems highlights the relevance of conformational dynamicity in mediating rapid ET.

Ligand Modifications Produce Two-Step Magnetic Switching in a Cobalt(dioxolene) Complex.

Mononuclear monodioxolene valence tautomeric (VT) cobalt complexes typically exist in their low spin (l.s.) CoIII (cat2- ) and high spin (h.s.) CoII (sq⋅- ) forms (cat2- =catecholato, and sq⋅- =seminquinonato forms of 3,5-di-t Bu-1,2-dioxolene), which reversibly interconvert via temperature-dependent intramolecular electron transfer. Typically, the remaining four coordination sites on cobalt are supported by a tetradentate ligand whose properties influence the temperature at which VT occurs. We report that replacing one chelating pyridyl arm of tris(2-pyridylmethyl)amine (tpa) with a weaker field ortho-anisole moiety facilitates access to a third magnetic state, and examine a series of related complexes. Variable temperature crystallographic, magnetic, calorimetric, and spectroscopic studies support that this third state is consistent with l.s. CoII (sq⋅- ). Thus, our ligand modifications not only provide access to the VT transition from l.s. CoIII (cat2- ) to l.s. CoII (sq⋅- ), but at higher temperatures, the complex undergoes spin crossover from l.s. CoII (sq⋅- ) to h.s. CoII (sq⋅- ), representing the first example of two-step magnetic switching in a mononuclear monodioxolene cobalt complex. We hypothesize that ligand dynamicity may facilitate access to the rarely observed l.s. CoII (sq⋅- ) intermediate state, suggesting a new design criterion in the development of stimulus-responsive multi-state molecular switches.

Conformational dynamicity in a copper(II) coordination complex.

The geometries of copper coordination complexes are intricately related to their electron transfer capabilities, but the role of dynamics in these processes are not fully understood. We have previously reported CuCl(dpaOMe), a complex exhibiting conformational fluxionality in its CuI state and rigidity upon oxidation to CuII. Here, we report the synthesis and characterization of [CuCl(dpaSMe)]+/0, a complex exhibiting relative rigidity in its CuI state and structural dynamics upon oxidation to CuII. The dynamics of [CuCl(dpaSMe)]+ were characterized via X-ray diffraction, cyclic voltammetry, and EPR spectroscopy, where temperature-dependent interconversion between trigonal bipyramidal and square pyramidal geometries is observed. Coupling these solid and solution-state characterization data enabled assignment of the coordination geometries involved. Factors impacting these dynamics and their potential implications for electron transfer are discussed.

Engineering a Conformationally Switchable Artificial Metalloprotein.

Many naturally occurring metalloenzymes are gated by rate-limiting conformational changes, and there exists a critical interplay between macroscopic structural rearrangements of the protein and subatomic changes affecting the electronic structure of embedded metallocofactors. Despite this connection, most artificial metalloproteins (ArMs) are prepared in structurally rigid protein hosts. To better model the natural mechanisms of metalloprotein reactivity, we have developed conformationally switchable ArMs (swArMs) that undergo a large-scale structural rearrangement upon allosteric effector binding. The swArMs reported here contain a Co(dmgH)2(X) cofactor (dmgH = dimethylglyoxime and X = N3-, H3C-, and iPr-). We used UV-vis absorbance and energy-dispersive X-ray fluorescence spectroscopies, along with protein assays, and mass spectrometry to show that these metallocofactors are installed site-specifically and stoichiometrically via direct Co-S cysteine ligation within the Escherichia coli glutamine binding protein (GlnBP). Structural characterization by single-crystal X-ray diffraction unveils the precise positioning and microenvironment of the metallocofactor within the protein fold. Fluorescence, circular dichroism, and infrared spectroscopies, along with isothermal titration calorimetry, reveal that allosteric Gln binding drives a large-scale protein conformational change. In swArMs containing a Co(dmgH)2(CH3) cofactor, we show that the protein stabilizes the otherwise labile Co-S bond relative to the free complex. Kinetics studies performed as a function of temperature and pH reveal that the protein conformational change accelerates this bond dissociation in a pH-dependent fashion. We present swArMs as a robust platform for investigating the interplay between allostery and metallocofactor regulation.

Toward Improved Charge Separation through Conformational Control in Copper Coordination Complexes.

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.

Conformationally dynamic copper coordination complexes.

The interplay between oxidation state and coordination geometry dictates both kinetic and thermodynamic properties underlying electron transfer events in copper coordination complexes. An ability to stabilize both CuI and CuII oxidation states in a single conformationally dynamic chelating ligand allows access to controlled redox reactivity. We report an analysis of the conformational dynamics of CuI complexes bearing dipicolylaniline (dpaR) ligands, with ortho-aniline substituents R = H and R = OMe. Variable temperature NMR spectroscopy and electrochemical experiments suggest that in solution at room temperature, an equilibrium exists between two conformers. Two metal-centered redox events are observed which, bolstered by structural information from single crystal X-ray diffraction and solution information from EPR and NMR spectroscopies, are ascribed to the CuII/I couple in planar and tetrahedral conformations. Activation and equilibrium parameters for these structural interconversions are presented and provide entry to leveraging redox-triggered conformational dynamics at Cu.

Intramolecular Hydrogen-Bond Interactions Tune Reactivity in Biomimetic Bis(µ-hydroxo)dicobalt Complexes.

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 Co2(µ-OH)2 diamond core stabilized by intramolecular H-bond interactions. We define the conditions required for the kinetically controlled synthesis of these complexes: [Co2(µ-OH)2(µ-OAc)(κ1-OAc)2(pyR)4][PF6] (1R), where OAc = acetate and pyR = pyridine with para-substituent R, and we describe a homologous series of 1R in which the para-R substituent on pyridine is modulated. The solid state X-ray diffraction (XRD) structures of 1R are similar across the series, but in solution, their 1H 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 κ1-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: [Co4(µ-O)4(µ3-OAc)4(pyR)4] (2R), 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 pKa in the presence of intramolecular H-bond interactions to maintain stability as the octahedral d6 centers become increasingly acidic. Nature may similarly employ dynamic carboxylate-mediated H-bond interactions to control the reactivity of acidic transition metal-oxo intermediates.

Subunit Interaction Dynamics of Class Ia Ribonucleotide Reductases: In Search of a Robust Assay.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides (NDP) to deoxynucleotides (dNDP), in part, by controlling the ratios and quantities of dNTPs available for DNA replication and repair. The active form of Escherichia coli class Ia RNR is an asymmetric α2ß2 complex in which α2 contains the active site and ß2 contains the stable diferric-tyrosyl radical cofactor responsible for initiating the reduction chemistry. Each dNDP is accompanied by disulfide bond formation. We now report that, under in vitro conditions, ß2 can initiate turnover in α2 catalytically under both "one" turnover (no external reductant, though producing two dCDPs) and multiple turnover (with an external reductant) assay conditions. In the absence of reductant, rapid chemical quench analysis of a reaction of α2, substrate, and effector with variable amounts of ß2 (1-, 10-, and 100-fold less than α2) yields 3 dCDP/α2 at all ratios of α2:ß2 with a rate constant of 8-9 s-1, associated with a rate-limiting conformational change. Stopped-flow fluorescence spectroscopy with a fluorophore-labeled ß reveals that the rate constants for subunit association (163 ± 7 µM-1 s-1) and dissociation (75 ± 10 s-1) are fast relative to turnover, consistent with catalytic ß2. When assaying in the presence of an external reducing system, the turnover number is dictated by the ratio of α2:ß2, their concentrations, and the concentration and nature of the reducing system; the rate-limiting step can change from the conformational gating to a step or steps involving disulfide rereduction, dissociation of the inhibited α4ß4 state, or both. The issues encountered with E. coli RNR are likely of importance in all class I RNRs and are central to understanding the development of screening assays for inhibitors of these enzymes.

Artificial Metalloproteins Containing Co4O4 Cubane Active Sites.

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 thin film 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.

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

The Escherichia coli class Ia ribonucleotide reductase (RNR) achieves forward and reverse proton-coupled electron transfer (PCET) over a pathway of redox active amino acids (ß-Y122 ⇌ ß-Y356 ⇌ α-Y731 ⇌ α-Y730 ⇌ α-C439) spanning ∼35 Å and two subunits every time it turns over. We have developed photoRNRs that allow radical transport to be phototriggered at tyrosine (Y) or fluorotyrosine (FnY) residues along the PCET pathway. We now report a new photoRNR in which photooxidation of a tryptophan (W) residue replacing Y356 within the α/ß subunit interface proceeds by a stepwise ET/PT (electron transfer then proton transfer) mechanism and provides an orthogonal spectroscopic handle with respect to radical pathway residues Y731 and Y730 in α. This construct displays an ∼3-fold enhancement in photochemical yield of W(•) relative to F3Y(•) and a ∼7-fold enhancement relative to Y(•). Photogeneration of the W(•) radical occurs with a rate constant of (4.4 ± 0.2) × 10(5) s(-1), which obeys a Marcus correlation for radical generation at the RNR subunit interface. Despite the fact that the Y → W variant displays no enzymatic activity in the absence of light, photogeneration of W(•) within the subunit interface results in 20% activity for turnover relative to wild-type RNR under the same conditions.

Charge-Transfer Dynamics at the α/ß 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 multistep proton-coupled electron transfer (PCET) over a pathway of redox active amino acids spanning ∼35 Å and two subunits (α2 and ß2). Despite the fact that PCET in RNR is rapid, slow conformational changes mask examination of the kinetics of these steps. As such, we have pioneered methodology in which site-specific incorporation of a [Re(I)] photooxidant on the surface of the ß2 subunit (photoß2) allows photochemical oxidation of the adjacent PCET pathway residue ß-Y356 and time-resolved spectroscopic observation of the ensuing reactivity. A series of photoß2s capable of performing photoinitiated substrate turnover have been prepared in which four different fluorotyrosines (FnYs) are incorporated in place of ß-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/ß 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 ß-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.

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 α2ß2 complex that undergoes reversible, long-range proton-coupled electron transfer (PCET) over a pathway of redox active amino acids (ß-Y122 → [ß-W48] → ß-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 [Re(I)] photooxidant site-specifically incorporated at position 355 ([Re]-ß2), adjacent to PCET pathway residue Y356 in ß. [Re]-ß2 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'-[(2)H]-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) × 10(4) 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.

Modulation of Y356 photooxidation in E. coli class Ia ribonucleotide reductase by Y731 across the α2:ß2 interface.

Substrate turnover in class Ia ribonucleotide reductase (RNR) requires reversible radical transport across two subunits over 35 Å, which occurs by a multistep proton-coupled electron-transfer mechanism. Using a photooxidant-labeled ß2 subunit of Escherichia coli class Ia RNR, we demonstrate photoinitiated oxidation of a tyrosine in an α2:ß2 complex, which results in substrate turnover. Using site-directed mutations of the redox-active tyrosines at the subunit interface, Y356F(ß) 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(ß)-Y731(α)-Y730(α) triad is crucial to radical transport in RNR.

Generation of a stable, aminotyrosyl radical-induced α2ß2 complex of Escherichia coli class Ia ribonucleotide reductase.

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•) in the RNR small (ß2) subunit over a 35-Å pathway of redox-active amino acids: Y122• ↔ [W48?] ↔ Y356 in ß2 to Y731 ↔ Y730 ↔ C439 in α2. When 3-aminotyrosine (NH2Y) is incorporated in place of Y730, a long-lived NH2Y730• is generated in α2 in the presence of wild-type (wt)-ß2, substrate, and effector. This radical intermediate is chemically and kinetically competent to generate dNDPs. Herein, evidence is presented that NH2Y730• induces formation of a kinetically stable α2ß2 complex. Under conditions that generate NH2Y730•, binding between Y730NH2Y-α2 and wt-ß2 is 25-fold tighter (Kd = 7 nM) than for wt-α2