Preparation and characterization of MnIIMnIII complexes with relevance to class Ib ribonucleotide reductases.

The Mn2 complex [MnII2(TPDP)(O2CPh)2](BPh4) (1, TPDP = 1,3-bis(bis(pyridin-2-ylmethyl)amino)propan-2-ol, Ph =phenyl) was prepared and subsequently characterized via single-crystal X-ray diffraction, X-ray absorption, electronic absorption, and infrared spectroscopies, and mass spectrometry. 1 was prepared in order to explore its properties as a structural and functional mimic of class Ib ribonucleotide reductases (RNRs). 1 reacted with superoxide anion (O2•-) to generate a peroxido-MnIIMnIII complex, 2. The electronic absorption and electron paramagnetic resonance (EPR) spectra of 2 were similar to previously published peroxido-MnIIMnIII species. Furthermore, X-ray near edge absorption structure (XANES) studies indicated the conversion of a MnII2 core in 1 to a MnIIMnIII state in 2. Treatment of 2 with para-toluenesulfonic acid (p-TsOH) resulted in the conversion to a new MnIIMnIII species, 3, rather than causing O-O bond scission, as previously encountered. 3 was characterized using electronic absorption, EPR, and X-ray absorption spectroscopies. Unlike other reported peroxido-MnIIMnIII species, 3 was capable of oxidative O-H activation, mirroring the generation of tyrosyl radical in class Ib RNRs, however without accessing the MnIIIMnIV state.

Isolation and characterization of a bis(dithiolene)-supported tungsten-acetylenic complex as a model for acetylene hydratase.

Acetylene hydratase is currently the only known mononuclear tungstoenzyme that does not catalyze a net redox reaction. The conversion of acetylene to acetaldehyde is proposed to occur at a W(IV) active site through first-sphere coordination of the acetylene substrate. To date, a handful of tungsten complexes have been shown to bind acetylene, but many lack the bis(dithiolene) motif of the native enzyme. The model compound, [W(O)(mnt)2]2-, where mnt2- is 1,2-dicyano-1,2-dithiolate, was previously reported to bind an electrophilic acetylene substrate, dimethyl acetylenedicarboxylate, and characterized by FT-IR, UV-vis, potentiometry, and mass spectrometry (Yadav, J; Das, S. K.; Sarkar, S., J. Am. Chem. Soc., 1997, 119, 4316-4317). By slightly changing the electrophilic acetylene substrate, an acetylenic-bis(dithiolene)­tungsten(IV) complex has been isolated and characterized by FT-IR, UV-vis, NMR, X-ray diffraction, and X-ray absorption spectroscopy. Activation parameters for complex formation were also determined and suggest coordination-sphere reorganization is a limiting factor in the model complex reactivity.

Evaluating Mass Spectrometry-Based Hydroxyl Radical Protein Footprinting of a Benchtop Flash Oxidation System against a Synchrotron X-ray Beamline.

Hydroxyl radical protein footprinting (HRPF) using synchrotron X-ray radiation (XFP) and mass spectrometry is a well-validated structural biology method that provides critical insights into macromolecular structural dynamics, such as determining binding sites, measuring affinity, and mapping epitopes. Numerous alternative sources for generating the hydroxyl radicals (•OH) needed for HRPF, such as laser photolysis and plasma irradiation, complement synchrotron-based HRPF, and a recently developed commercially available instrument based on flash lamp photolysis, the FOX system, enables access to laboratory benchtop HRPF. Here, we evaluate performing HRPF experiments in-house with a benchtop FOX instrument compared to synchrotron-based X-ray footprinting at the NSLS-II XFP beamline. Using lactate oxidase (LOx) as a model system, we carried out •OH labeling experiments using both instruments, followed by nanoLC-MS/MS bottom-up peptide mass mapping. Experiments were performed under high glucose concentrations to mimic the highly scavenging conditions present in biological buffers and human clinical samples, where less •OH are available for reaction with the biomolecule(s) of interest. The performance of the FOX and XFP HRPF methods was compared, and we found that tuning the •OH dosage enabled optimal labeling coverage for both setups under physiologically relevant highly scavenging conditions. Our study demonstrates the complementarity of FOX and XFP labeling approaches, demonstrating that benchtop instruments such as the FOX photolysis system can increase both the throughput and the accessibility of the HRPF technique.

Class Ib Ribonucleotide Reductases: Activation of a Peroxido-MnIIMnIII to Generate a Reactive Oxo-MnIIIMnIV Oxidant.

In the postulated catalytic cycle of class Ib Mn2 ribonucleotide reductases (RNRs), a MnII2 core is suggested to react with superoxide (O2·-) to generate peroxido-MnIIMnIII and oxo-MnIIIMnIV entities prior to proton-coupled electron transfer (PCET) oxidation of tyrosine. There is limited experimental support for this mechanism. We demonstrate that [MnII2(BPMP)(OAc)2](ClO4) (1, HBPMP = 2,6-bis[(bis(2 pyridylmethyl)amino)methyl]-4-methylphenol) was converted to peroxido-MnIIMnIII (2) in the presence of superoxide anion that converted to (µ-O)(µ-OH)MnIIIMnIV (3) via the addition of an H+-donor (p-TsOH) or (µ-O)2MnIIIMnIV (4) upon warming to room temperature. The physical properties of 3 and 4 were probed using UV-vis, EPR, X-ray absorption, and IR spectroscopies and mass spectrometry. Compounds 3 and 4 were capable of phenol oxidation to yield a phenoxyl radical via a concerted PCET oxidation, supporting the proposed mechanism of tyrosyl radical cofactor generation in RNRs. The synthetic models demonstrate that the postulated O2/Mn2/tyrosine activation mechanism in class Ib Mn2 RNRs is plausible and provides spectral insights into intermediates currently elusive in the native enzyme.

In vitro maturation of NiSOD reveals a role for cytoplasmic histidine in processing and metalation.

The importance of cellular low molecular weight ligands in metalloenzyme maturation is largely unexplored. Maturation of NiSOD requires post-translational N-terminal processing of the proenzyme, SodN, by its cognate protease, SodX. Here we provide evidence for the participation of L-histidine in the protease-dependent maturation of nickel-dependent superoxide dismutase (NiSOD) from Streptomyces coelicolor. In vitro studies using purified proteins cloned from S. coelicolor and overexpressed in E. coli support a model where a ternary complex formed between the substrate (SodN), the protease (SodX) and L-Histidine creates a novel Ni-binding site that is capable of the N-terminal processing of SodN and specifically incorporates Ni into the apo-NiSOD product. Thus, L-Histidine serves many of the functions associated with a metallochaperone or, conversely, eliminates the need for a metallochaperone in NiSOD maturation.

Intact mass spectrometry screening to optimize hydroxyl radical dose for protein footprinting.

Hydroxyl radical protein footprinting (HRPF) using synchrotron radiation is a well-validated method to assess protein structure in the native solution state. In this method, X-ray radiolysis of water generates hydroxyl radicals that can react with solvent accessible side chains of proteins, with mass spectrometry used to detect the resulting labeled products. An ideal footprinting dose provides sufficient labeling to measure the structure but not so much as to influence the results. The optimization of hydroxyl radical dose is typically performed using an indirect Alexa488 fluorescence assay sensitive to hydroxyl radical concentration, but full evaluation of the experiment's outcome relies upon bottom-up liquid chromatography mass spectrometry (LC-MS) measurements to directly determine sites and extent of oxidative labeling at the peptide and protein level. A direct evaluation of the extent of labeling to provide direct and absolute measurements of dose and "safe" dose ranges in terms of, for example, average numbers of labels per protein, would provide immediate feedback on experimental outcomes prior to embarking on detailed LC-MS analyses. To this end, we describe an approach to integrate intact MS screening of labeled samples immediately following exposure, along with metrics to quantify the extent of observed labeling from the intact mass spectra. Intact MS results on the model protein lysozyme were evaluated in the context of Alexa488 assay results and a bottom-up LC-MS analysis of the same samples. This approach provides a basis for placing delivered hydroxyl radical dose metrics on firmer technical grounds for synchrotron X-ray footprinting of proteins, with explicit parameters to increase the likelihood of a productive experimental outcome. Further, the method directs approaches to provide absolute and direct dosimetry for all types of labeling for protein footprinting.

Aromatic and aliphatic hydrocarbon hydroxylation via a formally NiIVO oxidant.

The reaction of (NMe4)2[NiII(LPh)(OAc)] (1[OAc], LPh = 2,2',2''-nitrilo-tris-(N-phenylacetamide); OAc = acetate) with 3-chloroperoxybenzoic acid (m-CPBA) resulted in the formation of a self-hydroxylated NiIII-phenolate complex, 2, where one of the phenyl groups of LPh underwent hydroxylation. 2 was characterised by UV-Vis, EPR, and XAS spectroscopies and ESI-MS. 2 decayed to yield a previously characterised NiII-phenolate complex, 3. We postulate that self-hydroxylation was mediated by a formally NiIVO oxidant, formed from the reaction of 1[OAc] with m-CPBA, which undergoes electrophilic aromatic substitution to yield 2. This is supported by an analysis of the kinetic and thermodynamic properties of the reaction of 1[OAc] with m-CPBA. Addition of exogenous hydrocarbon substrates intercepted the self-hydroxylation process, producing hydroxylated products, providing further support for the formally NiIVO entity. This study demonstrates that the reaction between NiII salts and m-CPBA can lead to potent metal-based oxidants, in contrast to recent studies demonstrating carboxyl radical is a radical free-chain reaction initiator in NiII/m-CPBA hydrocarbon oxidation catalysis.

Synthesis and Characterization of a Masked Terminal Nickel-Oxide Complex.

In exploring terminal nickel-oxo complexes, postulated to be the active oxidant in natural and non-natural oxidation reactions, we report the synthesis of the pseudo-trigonal bipyramidal NiII complexes (K)[NiII (LPh )(DMF)] (1[DMF]) and (NMe4 )2 [NiII (LPh )(OAc)] (1[OAc]) (LPh =2,2',2''-nitrilo-tris-(N-phenylacetamide); DMF=N,N-dimethylformamide; - OAc=acetate). Both complexes were characterized using NMR, FTIR, ESI-MS, and X-ray crystallography, showing the LPh ligand to bind in a tetradentate fashion, together with an ancillary donor. The reaction of 1[OAc] with peroxyphenyl acetic acid (PPAA) resulted in the formation of [(LPh )NiIII -O-H⋅⋅⋅OAc]2- , 2, that displays many of the characteristics of a terminal Ni=O species. 2 was characterized by UV-Vis, EPR, and XAS spectroscopies and ESI-MS. 2 decayed to yield a NiII -phenolate complex 3 (through aromatic electrophilic substitution) that was characterized by NMR, FTIR, ESI-MS, and X-ray crystallography. 2 was capable of hydroxylation of hydrocarbons and epoxidation of olefins, as well as oxygen atom transfer oxidation of phosphines at exceptional rates. While the oxo-wall remains standing, this complex represents an excellent example of a masked metal-oxide that displays all of the properties expected of the ever elusive terminal M=O beyond the oxo-wall.

An automated liquid jet for fluorescence dosimetry and microsecond radiolytic labeling of proteins.

X-ray radiolytic labeling uses broadband X-rays for in situ hydroxyl radical labeling to map protein interactions and conformation. High flux density beams are essential to overcome radical scavengers. However, conventional sample delivery environments, such as capillary flow, limit the use of a fully unattenuated focused broadband beam. An alternative is to use a liquid jet, and we have previously demonstrated that use of this form of sample delivery can increase labeling by tenfold at an unfocused X-ray source. Here we report the first use of a liquid jet for automated inline quantitative fluorescence dosage characterization and sample exposure at a high flux density microfocused synchrotron beamline. Our approach enables exposure times in single-digit microseconds while retaining a high level of side-chain labeling. This development significantly boosts the method's overall effectiveness and efficiency, generates high-quality data, and opens up the arena for high throughput and ultrafast time-resolved in situ hydroxyl radical labeling.

Multiplex Chemical Labeling of Amino Acids for Protein Footprinting Structure Assessment.

Protein footprinting with mass spectrometry is an established structural biology technique for mapping solvent accessibility and assessing molecular-level interactions of proteins. In hydroxyl radical protein footprinting (HRPF), hydroxyl (OH) radicals generated by water radiolysis or other methods covalently label protein side chains. Because of the wide dynamic range of OH reactivity, not all side chains are easily detected in a single experiment. Novel reagent development and the use of radical chain reactions for labeling, including trifluoromethyl radicals, is a potential approach to normalize the labeling across a diverse set of residues. HRPF in the presence of a trifluoromethylation reagent under the right conditions could provide a "one-pot" reaction for multiplex labeling of protein side chains. Toward this goal, we have systematically evaluated amino acid labeling with the recently investigated Langlois' reagent (LR) activated by X-ray-mediated water radiolysis, followed by three different mass spectrometry methods. We compared the reactivity of CF3 and OH radical labeling for all 20 protein side chains in a competition-free environment. We found that all 20 amino acids exhibited CF3 or OH labeling in LR. Our investigations provide the evidence and knowledge set to perfect hydroxyl radical-activated trifluoromethyl chemistry as "one-pot" reaction for multiplex labeling of protein side chains to achieve higher resolution in HRPF.

In vitro functional analysis of gRNA sites regulating assembly of hepatitis B virus.

The roles of RNA sequence/structure motifs, Packaging Signals (PSs), for regulating assembly of an HBV genome transcript have been investigated in an efficient in vitro assay containing only core protein (Cp) and RNA. Variants of three conserved PSs, within the genome of a strain not used previously, preventing correct presentation of a Cp-recognition loop motif are differentially deleterious for assembly of nucleocapsid-like particles (NCPs). Cryo-electron microscopy reconstruction of the T = 4 NCPs formed with the wild-type gRNA transcript, reveal that the interior of the Cp shell is in contact with lower resolution density, potentially encompassing the arginine-rich protein domains and gRNA. Symmetry relaxation followed by asymmetric reconstruction reveal that such contacts are made at every symmetry axis. We infer from their regulation of assembly that some of these contacts would involve gRNA PSs, and confirmed this by X-ray RNA footprinting. Mutation of the ε stem-loop in the gRNA, where polymerase binds in vivo, produces a poor RNA assembly substrate with Cp alone, largely due to alterations in its conformation. The results show that RNA PSs regulate assembly of HBV genomic transcripts in vitro, and therefore may play similar roles in vivo, in concert with other molecular factors.

New high-throughput endstation to accelerate the experimental optimization pipeline for synchrotron X-ray footprinting.

Synchrotron X-ray footprinting (XF) is a growing structural biology technique that leverages radiation-induced chemical modifications via X-ray radiolysis of water to produce hydroxyl radicals that probe changes in macromolecular structure and dynamics in solution states of interest. The X-ray Footprinting of Biological Materials (XFP) beamline at the National Synchrotron Light Source II provides the structural biology community with access to instrumentation and expert support in the XF method, and is also a platform for development of new technological capabilities in this field. The design and implementation of a new high-throughput endstation device based around use of a 96-well PCR plate form factor and supporting diagnostic instrumentation for synchrotron XF is described. This development enables a pipeline for rapid comprehensive screening of the influence of sample chemistry on hydroxyl radical dose using a convenient fluorescent assay, illustrated here with a study of 26 organic compounds. The new high-throughput endstation device and sample evaluation pipeline now available at the XFP beamline provide the worldwide structural biology community with a robust resource for carrying out well optimized synchrotron XF studies of challenging biological systems with complex sample compositions.

Formation of cobalt-oxygen intermediates by dioxygen activation at a mononuclear nonheme cobalt(ii) center.

A mononuclear nonheme cobalt(ii) complex, [(TMG3tren)CoII(OTf)](OTf) (1), activates dioxygen in the presence of hydrogen atom donor substrates, such as tetrahydrofuran and cyclohexene, resulting in the generation of a cobalt(ii)-alkylperoxide intermediate (2), which then converts to the previously reported cobalt(iv)-oxo complex, [(TMG3tren)CoIV(O)]2+-(Sc(OTf)3)n (3), in >90% yield upon addition of a redox-inactive metal ion, Sc(OTf)3. Intermediates 2 and 3 represent the cobalt analogues of the proposed iron(ii)-alkylperoxide precursor that converts to an iron(iv)-oxo intermediate via O-O bond heterolysis in pterin-dependent nonheme iron oxygenases. In reactivity studies, 2 shows an amphoteric reactivity in electrophilic and nucleophilic reactions, whereas 3 is an electrophilic oxidant. To the best of our knowledge, the present study reports the first example showing the generation of cobalt-oxygen intermediates by activating dioxygen at a cobalt(ii) center and the reactivities of the cobalt-oxygen intermediates in oxidation reaction.

Oxo-Free Hydrocarbon Oxidation by an Iron(III)-Isoporphyrin Complex.

Metal-halides that perform proton coupled electron-transfer (PCET) oxidation are an important new class of high-valent oxidant. In investigating metal-dihalides, we reacted [FeIII(Cl)(T(OMe)PP)] (1, T(OMe)PP = meso-tetra(4-methoxyphenyl)porphyrinyl) with (dichloroiodo)benzene. An FeIII-meso-chloro-isoporphyrin complex [FeIII(Cl)2(T(OMe)PP-Cl)] (2) was obtained. 2 was characterized by electronic absorption, 1H NMR, EPR, and X-ray absorption spectroscopies and mass spectrometry with support from computational analyses. 2 was reacted with a series of hydrocarbon substrates. The measured kinetic data exhibited a nonlinear behavior, whereby the oxidation followed a hydrogen-atom-transfer (HAT) PCET mechanism. The meso-chlorine atom was identified as the HAT agent. In one case, a halogenated product was identified by mass spectrometry. Our findings demonstrate that oxo-free hydrocarbon oxidation with heme systems is possible and show the potential for iron-dihalides in oxidative hydrocarbon halogenation.

Structural basis for carotenoid cleavage by an archaeal carotenoid dioxygenase.

Apocarotenoids are important signaling molecules generated from carotenoids through the action of carotenoid cleavage dioxygenases (CCDs). These enzymes have a remarkable ability to cleave carotenoids at specific alkene bonds while leaving chemically similar sites within the polyene intact. Although several bacterial and eukaryotic CCDs have been characterized, the long-standing goal of experimentally visualizing a CCD-carotenoid complex at high resolution to explain this exquisite regioselectivity remains unfulfilled. CCD genes are also present in some archaeal genomes, but the encoded enzymes remain uninvestigated. Here, we address this knowledge gap through analysis of a metazoan-like archaeal CCD from Candidatus Nitrosotalea devanaterra (NdCCD). NdCCD was active toward ß-apocarotenoids but did not cleave bicyclic carotenoids. It exhibited an unusual regiospecificity, cleaving apocarotenoids solely at the C14'-C13' alkene bond to produce ß-apo-14'-carotenals. The structure of NdCCD revealed a tapered active site cavity markedly different from the broad active site observed for the retinal-forming Synechocystis apocarotenoid oxygenase (SynACO) but similar to the vertebrate retinoid isomerase RPE65. The structure of NdCCD in complex with its apocarotenoid product demonstrated that the site of cleavage is defined by interactions along the substrate binding cleft as well as selective stabilization of reaction intermediates at the scissile alkene. These data on the molecular basis of CCD catalysis shed light on the origins of the varied catalytic activities found in metazoan CCDs, opening the possibility of modifying their activity through rational chemical or genetic approaches.

Protein Footprinting: Auxiliary Engine to Power the Structural Biology Revolution.

Structural biology is entering an exciting time where many new high-resolution structures of large complexes and membrane proteins are determined regularly. These advances have been driven by over fifteen years of technology advancements, first in macromolecular crystallography, and recently in Cryo-electron microscopy. These structures are allowing detailed questions about functional mechanisms of the structures, and the biology enabled by these structures, to be addressed for the first time. At the same time, mass spectrometry technologies for protein structure analysis, "footprinting" studies, have improved their sensitivity and resolution dramatically and can provide detailed sub-peptide and residue level information for validating structures and interactions or understanding the dynamics of structures in the context of ligand binding or assembly. In this perspective, we review the use of protein footprinting to extend our understanding of macromolecular systems, particularly for systems challenging for analysis by other techniques, such as intrinsically disordered proteins, amyloidogenic proteins, and other proteins/complexes so far recalcitrant to existing methods. We also illustrate how the availability of high-resolution structural information can be a foundation for a suite of hybrid approaches to divine structure-function relationships beyond what individual techniques can deliver.

High-Valent d7 NiIII versus d8 CuIII Oxidants in PCET.

Oxygenases have been postulated to utilize d4 FeIV and d8 CuIII oxidants in proton-coupled electron transfer (PCET) hydrocarbon oxidation. In order to explore the influence the metal ion and d-electron count can hold over the PCET reactivity, two metastable high-valent metal-oxygen adducts, [NiIII(OAc)(L)] (1b) and [CuIII(OAc)(L)] (2b), L = N,N'-(2,6-diisopropylphenyl)-2,6-pyridinedicarboxamidate, were prepared from their low-valent precursors [NiII(OAc)(L)]- (1a) and [CuII(OAc)(L)]- (2a). The complexes 1a/b-2a/b were characterized using nuclear magnetic resonance, Fourier transform infrared, electron paramagnetic resonance, X-ray diffraction, and absorption spectroscopies and mass spectrometry. Both complexes were capable of activating substrates through a concerted PCET mechanism (hydrogen atom transfer, HAT, or concerted proton and electron transfer, CPET). The reactivity of 1b and 2b toward a series of para-substituted 2,6-di-tert-butylphenols (p-X-2,6-DTBP; X = OCH3, C(CH3)3, CH3, H, Br, CN, NO2) was studied, showing similar rates of reaction for both complexes. In the oxidation of xanthene, the d8 CuIII oxidant displayed a small increase in the rate constant compared to that of the d7 NiIII oxidant. The d8 CuIII oxidant was capable of oxidizing a large family of hydrocarbon substrates with bond dissociation enthalpy (BDEC-H) values up to 90 kcal/mol. It was previously observed that exchanging the ancillary anionic donor ligand in such complexes resulted in a 20-fold enhancement in the rate constant, an observation that is further enforced by comparison of 1b and 2b to the literature precedents. In contrast, we observed only minor differences in the rate constants upon comparing 1b to 2b. It was thus concluded that in this case the metal ion has a minor impact, while the ancillary donor ligand yields more kinetic control over HAT/CPET oxidation.

Highly Reactive CoIII,IV2(µ-O)2 Diamond Core Complex That Cleaves C-H Bonds.

The selective activation of strong sp3 C-H bonds at mild conditions is a key step in many biological and synthetic transformations and an unsolved challenge for synthetic chemists. In nature, soluble methane monooxygenase (sMMO) is one representative example of nonheme dinuclear iron-dependent enzymes that activate strong sp3 C-H bonds by a high-valent diiron(IV) intermediate Q. To date, synthetic model complexes of sMMO-Q have shown limited abilities to oxidize strong C-H bonds. In this work, we generated a high-valent CoIII,IV2(µ-O)2 complex 3 supported by a tetradentate tris(2-pyridylmethyl)amine (TPA) ligand via one-electron oxidation of its CoIII2(µ-O)2 precursor 2. Characterization of 2 and 3 using X-ray absorption spectroscopy and DFT calculations showed that both species possess a diamond core structure with a short Co···Co distance of 2.78 Å. Furthermore, 3 is an EPR active species showing an S = 1/2 signal with clearly observable hyperfine splittings originated from the coupling of the 59Co nuclear spin with the electronic spin. Importantly, 3 is a highly reactive oxidant for sp3 C-H bonds, and an oxygenation reagent. 3 has the highest rate constant (1.5 M-1 s-1 at -60 °C) for oxidizing 9,10-dihydroanthracene (DHA) compared to diamond core complexes of other first-row transition metals including Mn, Fe and Cu reported previously. Specifically, 3 is about 4-5 orders of magnitude more reactive than the diiron analogs FeIII,IV2(µ-O)2 and FeIV2(µ-O)2 supported by TPA and related ligands. These findings shed light on future development of more reactive approaches for C-H bond activation by bioinspired dicobalt complexes.

The XFP (17-BM) beamline for X-ray footprinting at NSLS-II.

Hydroxyl-radical mediated synchrotron X-ray footprinting (XF) is a powerful solution-state technique in structural biology for the study of macromolecular structure and dynamics of proteins and nucleic acids, with several synchrotron resources available to serve the XF community worldwide. The XFP (Biological X-ray Footprinting) beamline at the NSLS-II was constructed on a three-pole wiggler source at 17-BM to serve as the premier beamline for performing this technique, providing an unparalleled combination of high flux density broadband beam, flexibility in beam morphology, and sample handling capabilities specifically designed for XF experiments. The details of beamline design, beam measurements, and science commissioning results for a standard protein using the two distinct XFP endstations are presented here. XFP took first light in 2016 and is now available for general user operations through peer-reviewed proposals. Currently, beam sizes from 450 µm × 120 µm to 2.7 mm × 2.7 mm (FWHM) are available, with a flux of 1.6 × 1016 photons s-1 (measured at 325 mA ring current) in a broadband (∼5-16 keV) beam. This flux is expected to rise to 2.5 × 1016 photons s-1 at the full NSLS-II design current of 500 mA, providing an incident power density of >500 W mm-2 at full focus.

Redesign of a Copper Storage Protein into an Artificial Hydrogenase.

We report the construction of an artificial hydrogenase (ArH) by reengineering a Cu storage protein (Cspl) into a Ni-binding protein (NBP) employing rational metalloprotein design. The hypothesis driven design approach involved deleting existing Cu sites of Csp1 and identification of a target tetrathiolate Ni binding site within the protein scaffold followed by repacking the hydrophobic core. Guided by modeling, the NBP was expressed and purified in high purity. NBP is a well-folded and stable construct displaying native-like unfolding behavior. Spectroscopic and computational studies indicated that the NBP bound nickel in a distorted square planar geometry that validated the design. Ni(II)-NBP is active for photo-induced H2 evolution following a reductive quenching mechanism. Ni(II)-NBP catalyzed H+ reduction to H2 gas electrochemically as well. Analysis of the catalytic voltammograms established a proton-coupled electron transfer (PCET) mechanism. Electrolysis studies confirmed H2 evolution with quantitative Faradaic yields. Our studies demonstrate an important scope of rational metalloprotein design that allows imparting functions into protein scaffolds that have natively not evolved to possess the same function of the target metalloprotein constructs.