Oxygen Isotopologues Resolved from Water Oxidation Electrocatalysis by Electron Paramagnetic Resonance Spectroscopy.

Electrocatalytic water oxidation is a key transformation in many strategies designed to harness solar energy and store it as chemical fuels. Understanding the mechanism(s) of the best electrocatalysts for water oxidation has been a fundamental chemical challenge for decades. Here, we quantitate evolved dioxygen isotopologue composition via gas-phase EPR spectroscopy to elucidate the mechanisms of water oxidation on metal oxide electrocatalysts with high precision. Isotope fractionation is paired with computational and kinetic modeling, showing that this technique is sensitive enough to differentiate O-O bond-forming steps. Strong agreement between experiment and theory indicates that for the nickel-iron layered double hydroxide─one of the best earth-abundant electrocatalysts to be studied─water oxidation proceeds via a dioxo coupling mechanism to form a side-bound peroxide rather than a hydroxide attack to form an end-bound peroxide.

Copper(II) coordination to the intrinsically disordered region of SARS-CoV-2 Nsp1.

The intrinsically disordered C-terminal peptide region of severe acute respiratory syndrome coronavirus 2 nonstructural protein-1 (Nsp1-CT) inhibits host protein synthesis by blocking messenger RNA (mRNA) access to the 40S ribosome entrance tunnel. Aqueous copper(II) ions bind to the disordered peptide with micromolar affinity, creating a possible strategy to restore protein synthesis during host infection. Electron paramagnetic resonance (EPR) and tryptophan fluorescence measurements on a 10-residue model of the disordered protein region (Nsp1-CT10), combined with advanced quantum mechanics calculations, suggest that the peptide binds to copper(II) as a multidentate ligand. Two optimized computational models of the copper(II)-peptide complexes were derived: One corresponding to pH 6.5 and the other describing the complex at pH 7.5 to 8.5. Simulated EPR spectra based on the calculated model structures are in good agreement with experimental spectra.

Intermolecular Proton-Coupled Electron Transfer Reactivity from a Persistent Charge-Transfer State for Reductive Photoelectrocatalysis.

Interest in applying proton-coupled electron transfer (PCET) reagents in reductive electro- and photocatalysis requires strategies that mitigate the competing hydrogen evolution reaction. Photoexcitation of a PCET donor to a charge-separated state (CSS) can produce a powerful H-atom donor capable of being electrochemically recycled at a comparatively anodic potential corresponding to its ground state. However, the challenge is designing a mediator with a sufficiently long-lived excited state for bimolecular reactivity. Here, we describe a powerful ferrocene-derived photoelectrochemical PCET mediator exhibiting an unusually long-lived CSS (τ ∼ 0.9 µs). In addition to detailed photophysical studies, proof-of-concept stoichiometric and catalytic proton-coupled reductive transformations are presented, which illustrate the promise of this approach.

Tryptophan extends the life of cytochrome P450.

Powerfully oxidizing enzymes need protective mechanisms to prevent self-destruction. The flavocytochrome P450 BM3 from Priestia megaterium (P450BM3) is a self-sufficient monooxygenase that hydroxylates fatty acid substrates using O2 and NADPH as co-substrates. Hydroxylation of long-chain fatty acids (≥C14) is well coupled to O2 and NADPH consumption, but shorter chains (≤C12) are more poorly coupled. Hydroxylation of p-nitrophenoxydodecanoic acid by P450BM3 produces a spectrophotometrically detectable product wherein the coupling of NADPH consumption to product formation is just 10%. Moreover, the rate of NADPH consumption is 1.8 times that of O2 consumption, indicating that an oxidase uncoupling pathway is operative. Measurements of the total number of enzyme turnovers before inactivation (TTN) indicate that higher NADPH concentrations increase TTN. At lower NADPH levels, added ascorbate increases TTN, while a W96H mutation leads to a decrease. The W96 residue is about 7 Å from the P450BM3 heme and serves as a gateway residue in a tryptophan/tyrosine (W/Y) hole transport chain from the heme to a surface tyrosine residue. The data indicate that two oxidase pathways protect the enzyme from damage by intercepting the powerfully oxidizing enzyme intermediate (Compound I) and returning it to its resting state. At high NADPH concentrations, reducing equivalents from the flavoprotein are delivered to Compound I by the usual reductase pathway. When NADPH is not abundant, however, oxidizing equivalents from Compound I can traverse a W/Y chain, arriving at the enzyme surface where they are scavenged by reductants. Ubiquitous tryptophan/tyrosine chains in highly oxidizing enzymes likely perform similar protective functions.

Resonance Raman spectra of blue copper proteins: Variable temperature spectra of Thermus thermophilus HB27 laccase.

The resonance Raman (rR) spectra of the oxidized type 1 copper active site (CuT1) in Thermus thermophilus HB27 laccase (Tth-lac) has been determined in the 20 to 80 °C temperature range using 633-nm excitation. The positions and relative intensities of rR peaks are virtually independent of temperature, indicating that CuT1 ligation is robust over the investigated range. The intensity-weighted average of Tth-lac Cu-SCys vibrations (<ν(Cu-SCys)>) = 423 cm-1) is higher than those of most cupredoxins but is comparable to those of other multicopper oxidases (MCOs). <ν(Cu-SCys)> values for Tth-lac and several CuT1 centers in cupredoxins and MCOs do not correlate well with Cu-SCys bond lengths but do exhibit systematic trends with redox thermodynamic properties. PROLOGUE: F. Ann Walker was a great scholar and dear friend. While at Columbia in the early 1960s, I (HBG) followed her graduate work at Brown on the effects of axial ligands on vanadyl ion EPR spectra. Dick Carlin, her thesis adviser, invited me to serve as external member of her thesis committee. I joined, made my way to Providence, met her just before the exam, and greatly admired (enjoyed!) her thoughtful responses to questions from physical chemists about metal-oxo electronic structures. Our friendship grew stronger over the years, enhanced by lively discussions of heme protein chemistry in San Francisco, Pasadena, Tucson, and at Gordon Research Conferences. Ann was a superstar in biological inorganic chemistry. She will be sorely missed but not forgotten.

Electronic Structures and Photoredox Chemistry of Tungsten(0) Arylisocyanides.

ConspectusThe high energy barriers associated with the reaction chemistry of inert substrates can be overcome by employing redox-active photocatalysts. Research in this area has grown exponentially over the past decade, as transition metal photosensitizers have been shown to mediate challenging organic transformations. Critical for the advancement of photoredox catalysis is the discovery, development, and study of complexes based on earth-abundant metals that can replace and/or complement established noble-metal-based photosensitizers.Recent work has focused on redox-active complexes of 3d metals, as photosensitizers containing these metals most likely would be scalable. Although low lying spin doublet ("spin flip") excited states of chromium(III) and metal-to-ligand charge transfer (MLCT) excited states of copper(I) have relatively long lifetimes, the electronic excited states of many other 3d metal complexes fall on dissociative potential energy surfaces, owing to the population of highly energetic σ-antibonding orbitals. Indeed, we and other investigators have shown that low lying spin singlet and triplet excited states of robust closed-shell metal complexes are too short-lived at room temperature to engage in bimolecular reactions in solutions. In principle, this problem could be overcome by designing and constructing 3d metal complexes containing strong field π-acceptor ligands, where thermally equilibrated MLCT or intraligand charge transfer excited states might fall well below the upper surfaces of dissociative 3d-3d states. Notably, such design elements have been exploited by investigators in very recent work on redox-active iron(II) systems. Another approach, one we have actively pursued, is to design and construct closed-shell complexes of earth-abundant 5d metals containing very strong π-acceptor ligands, where vertical excitation of 5d-5d excited states at the ground state geometry would require energies far above minima in the potential surfaces of MLCT excited states. As this requirement is met by tungsten(0) arylisocyanides, these complexes have been the focus of our work aimed at the development of robust redox-active photosensitizers.In the following Account, we review recent work on homoleptic tungsten(0) arylisocyanides. Originally reported by our group 45 years ago, W(CNAr)6 complexes have exceptionally large one- and two-photon absorption cross-sections. One- or two-photon excitation produces relatively long-lived (hundreds of nanoseconds to microsecond) MLCT excited states in high yields. These MLCT excited states, which are very strong reductants with E°(W+/*W0) = -2.2 to -3.0 V vs Fc[+/0], mediate photocatalysis of organic reactions with both visible and near-infrared (NIR) light. Here, we highlight design principles that led to the development of three generations of W(CNAr)6 photosensitizers; and we discuss likely steps in the mechanism of a prototypal W(CNAr)6-catalyzed base-promoted homolytic aromatic substitution reaction. Among the many potential applications of these very bright luminophores, two-photon imaging and two-photon-initiated polymerization are ones we plan to pursue.

Boronated Cyanometallates.

Thirteen boronated cyanometallates [M(CN-BR3)6]3/4/5- [M = Cr, Mn, Fe, Ru, Os; BR3 = BPh3, B(2,4,6,-F3C6H2)3, B(C6F5)3] and one metalloboratonitrile [Cr(NC-BPh3)6]3- have been characterized by X-ray crystallography and spectroscopy [UV-vis-near-IR, NMR, IR, spectroelectrochemistry, and magnetic circular dichroism (MCD)]; CASSCF+NEVPT2 methods were employed in calculations of electronic structures. For (t2g)5 electronic configurations, the lowest-energy ligand-to-metal charge-transfer (LMCT) absorptions and MCD C-terms in the spectra of boronated species have been assigned to transitions from cyanide π + B-C borane σ orbitals. CASSCF+NEVPT2 calculations including t1u and t2u orbitals reproduced t1u/t2u → t2g excitation energies. Many [M(CN-BR3)6]3/4- complexes exhibited highly electrochemically reversible redox couples. Notably, the reduction formal potentials of all five [M(CN-B(C6F5)3)6]3- anions scale with the LMCT energies, and Mn(I) and Cr(II) compounds, [K(18-crown-6)]5[Mn(CN-B(C6F5)3)6] and [K(18-crown-6)]4[Cr(CN-B(C6F5)3)6], are surprisingly stable. Continuous-wave and pulsed electron paramagnetic resonance (EPR; hyperfine sublevel correlation) spectra were collected for all Cr(III) complexes; as expected, 14N hyperfine splittings are greater for (Ph4As)3[Cr(NC-BPh3)6] than for (Ph4As)3[Cr(CN-BPh3)6].

Tryptophan-96 in cytochrome P450 BM3 plays a key role in enzyme survival.

Flavocytochrome P450 from Bacillus megaterium (P450BM3 ) is a natural fusion protein containing reductase and heme domains. In the presence of NADPH and dioxygen the enzyme catalyses the hydroxylation of long-chain fatty acids. Analysis of the P450BM3 structure reveals chains of closely spaced tryptophan and tyrosine residues that might serve as pathways for high-potential oxidizing equivalents to escape from the heme active site when substrate oxidation is not possible. Our investigations of the total number of enzyme turnovers before deactivation have revealed that replacement of selected tryptophan and tyrosine residues with redox inactive groups leads to a twofold reduction in enzyme survival time. Tryptophan-96 is critical for prolonging enzyme activity, suggesting a key protective role for this residue.

Does Tyrosine Protect S. coelicolor Laccase from Oxidative Degradation or Act as an Extended Catalytic Site?

We have investigated the roles of tyrosine (Tyr) and tryptophan (Trp) residues in the four-electron reduction of oxygen catalyzed by Streptomyces coelicolor laccase (SLAC). During normal enzymatic turnover in laccases, reducing equivalents are delivered to a type 1 Cu center (CuT1) and then are transferred over 13 Što a trinuclear Cu site (TNC: (CuT3)2CuT2) where O2 reduction occurs. The TNC in SLAC is surrounded by a large cluster of Tyr and Trp residues that can provide reducing equivalents when the normal flow of electrons is disrupted. Prior studies by Canters and co-workers [J. Am. Chem. Soc. 2009, 131 (33), 11680-11682] have shown that when O2 reacts with a reduced SLAC variant lacking the CuT1 center, a Tyr108• radical near the TNC forms rapidly. We have found that the Tyr108• radical is reduced 10 times faster than CuT12+ by excess ascorbate, possibly because of radical transfer along Tyr/Trp chains.

Surface cysteines could protect the SARS-CoV-2 main protease from oxidative damage.

The SARS-CoV-2 main protease (Mpro) is responsible for cleaving twelve nonstructural proteins from the viral polyprotein. Mpro, a cysteine protease, is characterized by a large number of noncatalytic cysteine (Cys) residues, none involved in disulfide bonds. In the absence of a tertiary-structure stabilizing role for these residues, a possible alternative is that they are involved in redox processes. We report experimental work in support of a proposal that surface cysteines on Mpro can protect the active-site Cys145 from oxidation by reactive oxygen species (ROS). In investigations of enzyme kinetics, we found that mutating three surface cysteines to serines did not greatly affect activity, which in turn indicates that these cysteines could protect Cys145 from oxidative damage.

Copper(II) Binding to the Intrinsically Disordered C-Terminal Peptide of SARS-CoV-2 Virulence Factor Nsp1.

The first encoded SARS-CoV-2 protein (Nsp1) binds to the human 40S ribosome and blocks synthesis of host proteins, thereby inhibiting critical elements of the innate immune response. The final 33 residues of the natively unstructured Nsp1 C-terminus adopt a helix-turn-helix geometry upon binding to the ribosome. We have characterized the fluctuating conformations of this peptide using circular dichroism spectroscopy along with measurements of tryptophan fluorescence and energy transfer. Tryptophan fluorescence decay kinetics reveal that copper(II) binds to the peptide at micromolar concentrations, and electron paramagnetic resonance spectroscopy indicates that the metal ion coordinates to the lone histidine residue.

Photoredox Catalysis Mediated by Tungsten(0) Arylisocyanides in 1,2-Difluorobenzene.

We have studied the photochemical cyclization of 1-(2-iodobenzyl)-pyrrole (IBP) and 1-(2-bromobenzyl)-pyrrole (BBP) to 5H-pyrrolo[2,1-a]isoindol catalyzed by W(CNDipp)6 (CNDipp = 2,6-diisopropylphenylisocyanide) in 1,2-difluorobenzene (DFB). Irradiation (445 nm) of W(CNDipp)6 (5 mol %) in DFB solution converted 78% of IBP (50 mM) to product after 1 h (16 turnovers). Addition of tetra-n-butyl ammonium hexafluorophosphate (TBAPF6) (0.2 M) to the DFB solution led to rapid photoinduced disappearance of W(CNDipp)6 but, remarkably, did not inhibit photochemical cyclization of IBP, indicating that IBP cyclization could be driven by a nonluminescent photocatalyst.

Photoredox Catalysis Mediated by Tungsten(0) Arylisocyanides.

W(CNAr)6 (CNAr = arylisocyanide) photoreductants catalyze base-promoted homolytic aromatic substitution (BHAS) of 1-(2-iodobenzyl)-pyrrole in deuterated benzene. Moderate to high efficiencies correlate with W(CNAr)6 excited-state reduction potentials upon one-photon 445 nm excitation, with 10 mol % loading of the most powerful photoreductants W(CNDipp)6 (CNDipp = 2,6-diisopropylphenylisocyanide) and W(CNDippPhOMe3)6 (CNDippPhOMe3 = 4-(3,4,5-trimethoxyphenyl)-2,6-diisopropylphenylisocyanide) affording nearly complete conversion. Stern-Volmer quenching experiments indicated that catalysis is triggered by substrate reductive dehalogenation. Taking advantage of the large two-photon absorption (TPA) cross sections of W(CNAr)6 complexes, we found that photocatalysis can be driven with femtosecond-pulsed 810 nm excitation. For both one- and two-photon excitation, photocatalysis was terminated by the formation of seven-coordinate WII-diiodo [WI2(CNAr)5] complexes. Notably, we discovered that W(CNDipp)6 can be regenerated by chemical reduction of WI2(CNDipp)5 with excess ligand present in solution.

Functional and protective hole hopping in metalloenzymes.

Electrons can tunnel through proteins in microseconds with a modest release of free energy over distances in the 15 to 20 Å range. To span greater distances, or to move faster, multiple charge transfers (hops) are required. When one of the reactants is a strong oxidant, it is convenient to consider the movement of a positively charged "hole" in a direction opposite to that of the electron. Hole hopping along chains of tryptophan (Trp) and tyrosine (Tyr) residues is a critical function in several metalloenzymes that generate high-potential intermediates by reactions with O2 or H2O2, or by activation with visible light. Examination of the protein structural database revealed that Tyr/Trp chains are common protein structural elements, particularly among enzymes that react with O2 and H2O2. In many cases these chains may serve a protective role in metalloenzymes by deactivating high-potential reactive intermediates formed in uncoupled catalytic turnover.

Photoinduced hole hopping through tryptophans in proteins.

Hole hopping through tryptophan/tyrosine chains enables rapid unidirectional charge transport over long distances. We have elucidated structural and dynamical factors controlling hopping speed and efficiency in two modified azurin constructs that include a rhenium(I) sensitizer, Re(His)(CO)3(dmp)+, and one or two tryptophans (W1, W2). Experimental kinetics investigations showed that the two closely spaced (3 to 4 Å) intervening tryptophans dramatically accelerated long-range electron transfer (ET) from CuI to the photoexcited sensitizer. In our theoretical work, we found that time-dependent density-functional theory (TDDFT) quantum mechanics/molecular mechanics/molecular dynamics (QM/MM/MD) trajectories of low-lying triplet excited states of ReI(His)(CO)3(dmp)+-W1(-W2) exhibited crossings between sensitizer-localized (*Re) and charge-separated [ReI(His)(CO)3(dmp•-)/(W1•+ or W2•+)] (CS1 or CS2) states. Our analysis revealed that the distances, angles, and mutual orientations of ET-active cofactors fluctuate in a relatively narrow range in which the cofactors are strongly coupled, enabling adiabatic ET. Water-dominated electrostatic field fluctuations bring *Re and CS1 states to a crossing where *Re(CO)3(dmp)+←W1 ET occurs, and CS1 becomes the lowest triplet state. ET is promoted by solvation dynamics around *Re(CO)3(dmp)+(W1); and CS1 is stabilized by Re(dmp•-)/W1•+ electron/hole interaction and enhanced W1•+ solvation. The second hop, W1•+←W2, is facilitated by water fluctuations near the W1/W2 unit, taking place when the electrostatic potential at W2 drops well below that at W1•+ Insufficient solvation and reorganization around W2 make W1•+←W2 ET endergonic, shifting the equilibrium toward W1•+ and decreasing the charge-separation yield. We suggest that multiscale TDDFT/MM/MD is a suitable technique to model the simultaneous evolution of photogenerated excited-state manifolds.

Role of intramolecular hydrogen bonds in promoting electron flow through amino acid and oligopeptide conjugates.

Elucidating the factors that control charge transfer rates in relatively flexible conjugates is of importance for understanding energy flows in biology as well as assisting the design and construction of electronic devices. Here, we report ultrafast electron transfer (ET) and hole transfer (HT) between a corrole (Cor) donor linked to a perylene-diimide (PDI) acceptor by a tetrameric alanine (Ala)4 Selective photoexcitation of the donor and acceptor triggers subpicosecond and picosecond ET and HT. Replacement of the (Ala)4 linker with either a single alanine or phenylalanine does not substantially affect the ET and HT kinetics. We infer that electronic coupling in these reactions is not mediated by tetrapeptide backbone nor by direct donor-acceptor interactions. Employing a combination of NMR, circular dichroism, and computational studies, we show that intramolecular hydrogen bonding brings the donor and the acceptor into proximity in a "scorpion-shaped" molecular architecture, thereby accounting for the unusually high ET and HT rates. Photoinduced charge transfer relies on a (Cor)NH…O=C-NH…O=C(PDI) electronic-coupling pathway involving two pivotal hydrogen bonds and a central amide group as a mediator. Our work provides guidelines for construction of effective donor-acceptor assemblies linked by long flexible bridges as well as insights into structural motifs for mediating ET and HT in proteins.

Third-Generation W(CNAr)6 Photoreductants (CNAr = Fused-Ring and Alkynyl-Bridged Arylisocyanides).

Homoleptic tungsten(0) arylisocyanides possess photophysical and photochemical properties that rival those of archetypal ruthenium(II) and iridium(III) polypyridine complexes. Previous studies established that extending the π-system of 2,6-diisopropylphenylisocyanide (CNDipp) by coupling aryl substituents para to the isocyanide functionality results in W(CNDippAr)6 oligoarylisocyanide complexes with greatly enhanced metal-to-ligand charge transfer (MLCT) excited-state properties relative to those of W(CNDipp)6. Extending electronic modifications to delineate additional design principles for this class of photosensitizers, herein we report a series of W(CNAr)6 compounds with naphthalene-based fused-ring (CN-1-(2-iPr)-Naph) and CNDipp-based alkynyl-bridged (CNDippCCAr) arylisocyanide ligands. Systematic variation of the secondary aromatic system in the CNDippCCAr platform provides a straightforward method to modulate the photophysical properties of W(CNDippCCAr)6 complexes, allowing access to an extended range of absorption/luminescence profiles and highly reducing excited states, while maintaining the high molar absorptivity MLCT absorption bands, high photoluminescence quantum yields, and long excited-state lifetimes of previous W(CNAr)6 complexes. Notably, W(CN-1-(2-iPr)-Naph)6 exhibits the longest excited-state lifetime of all W(CNAr)6 complexes explored thus far, highlighting the potential benefits of utilizing fused-ring arylisocyanide ligands in the construction of tungsten(0) photoreductants.

A Super-Oxidized Radical Cationic Icosahedral Boron Cluster.

While the icosahedral closo-[B12H12]2- cluster does not display reversible electrochemical behavior, perfunctionalization of this species via substitution of all 12 B-H vertices with alkoxy or benzyloxy (OR) substituents engenders reversible redox chemistry, providing access to clusters in the dianionic, monoanionic, and neutral forms. Here, we evaluated the electrochemical behavior of the electron-rich B12(O-3-methylbutyl)12 (1) cluster and discovered that a new reversible redox event that gives rise to a fourth electronic state is accessible through one-electron oxidation of the neutral species. Chemical oxidation of 1 with [N(2,4-Br2C6H3)3]•+ afforded the isolable [1]•+ cluster, which is the first example of an open-shell cationic B12 cluster in which the unpaired electron is proposed to be delocalized throughout the boron cluster core. The oxidation of 1 is also chemically reversible, where treatment of [1]•+ with ferrocene resulted in its reduction back to 1. The identity of [1]•+ is supported by EPR, UV-vis, multinuclear NMR (1H, 11B), and X-ray photoelectron spectroscopic characterization.

Hole Hopping through Cytochrome P450.

High-potential iron-oxo species are intermediates in the catalytic cycles of oxygenase enzymes. They can cause heme degradation and irreversible oxidation of nearby amino acids. We have proposed that there are protective mechanisms in which hole hopping from oxidized hemes through tryptophan/tyrosine chains generates a surface-exposed amino-acid oxidant that could be rapidly disarmed by reaction with cellular reductants. In investigations of cytochrome P450BM3, we identified Trp96 as a critical residue that could play such a protective role. This Trp is cation-π paired with Arg398 in 81% of mammalian P450s. Here we report on the effect of the Trp/Arg cation-π interaction on Trp96 formal potentials as well as on electronic coupling strengths between Trp96 and the heme both for wild type cytochrome P450 and selected mutants. Mutation of Arg398 to His, which decreases the Trp96 formal potential, increases Trp-heme electronic coupling; however, surprisingly, the rate of phototriggered electron transfer from a Ru-sensitizer (through Trp96) to the P450BM3 heme was unaffected by the Arg398His mutation. We conclude that Trp96 has moved away from Arg398, suggesting that the protective mechanism for P450s with this Trp-Arg pair is conformationally gated.

Stability/activity tradeoffs in Thermusthermophilus HB27 laccase.

We report the temperature dependence of the formal potential of type 1 copper (CuT1) in Thermusthermophilus HB27 laccase. Employing [Ru(NH3)4(bpy)](PF6)2 (0.505 vs. NHE) as the redox titrant, we found that the CuT12+/+ potential decreased from approximately 480 to 420 mV (vs. NHE) as the temperature was raised from 20 to 65 °C. Of importance is that the ΔSrc° of - 120 J mol-1 K-1 is substantially more negative than those for other blue copper proteins. We suggest that the highly unfavorable reduction entropy is attributable to CuT1 inaccessibility to the aqueous medium. Although the active site residues are buried, which is critical for maintaining thermostability, the flexibility around CuT1 is maintained, allowing enzyme activity at ambient temperature.