Highly efficient electrocatalytic CO2 reduction by a CrIII quaterpyridine complex.

Design tactics and mechanistic studies both remain as fundamental challenges during the exploitations of earth-abundant molecular electrocatalysts for CO2 reduction, especially for the rarely studied Cr-based ones. Herein, a quaterpyridyl CrIII catalyst is found to be highly active for CO2 electroreduction to CO with 99.8% Faradaic efficiency in DMF/phenol medium. A nearly one order of magnitude higher turnover frequency (86.6 s-1) over the documented Cr-based catalysts (<10 s-1) can be achieved at an applied overpotential of only 190 mV which is generally 300 mV lower than these precedents. Such a high performance at this low driving force originates from the metal-ligand cooperativity that stabilizes the low-valent intermediates and serves as an efficient electron reservoir. Moreover, a synergy of electrochemistry, spectroelectrochemistry, electron paramagnetic resonance, and quantum chemical calculations allows to characterize the key CrII, CrI, Cr0, and CO-bound Cr0 intermediates as well as to verify the catalytic mechanism.

Operando studies of Mn oxide based electrocatalysts for the oxygen evolution reaction.

Inspired by photosystem II (PS II), Mn oxide based electrocatalysts have been repeatedly investigated as catalysts for the electrochemical oxygen evolution reaction (OER), the anodic reaction in water electrolysis. However, a comparison of the conditions in biological OER catalysed by the water splitting complex CaMn4Ox with the requirements for an electrocatalyst for industrially relevant applications reveals fundamental differences. Thus, a systematic development of artificial Mn-based OER catalysts requires both a fundamental understanding of the catalytic mechanisms as well as an evaluation of the practicality of the system for industrial scale applications. Experimentally, both aspects can be approached using in situ and operando methods including spectroscopy. This paper highlights some of the major challenges common to different operando investigation methods and recent insights gained with them. To this end, vibrational spectroscopy, especially Raman spectroscopy, absorption techniques in the bandgap region and operando X-ray spectroelectrochemistry (SEC), both in the hard and soft X-ray regime are particularly focused on here. Technical challenges specific to each method are discussed first, followed by challenges that are specific to Mn oxide based systems. Finally, recent in situ and operando studies are reviewed. This analysis shows that despite the technical and Mn specific challenges, three specific key features are common to most of the studied systems with significant OER activity: structural disorder, Mn oxidation states between III and IV, and the appearance of layered birnessite phases in the active regime.

Enzymatic X-ray absorption spectroelectrochemistry.

The ability to observe the changes that occur at an enzyme active site during electrocatalysis can provide very valuable information for understanding the mechanism and ultimately aid in catalyst design. Herein, we discuss the development of X-ray absorption spectroscopy (XAS) in combination with electrochemistry for operando studies of enzymatic systems. XAS has had a long history of enabling geometric and electronic structural insights into the catalytic active sites of enzymes, however, XAS combined with electrochemistry (XA-SEC) has been exceedingly rare in bioinorganic applications. Herein, we discuss the challenges and opportunities of applying operando XAS to enzymatic electrocatalysts. The challenges due to the low concentration of the photoabsorber and the instability of the protein in the X-ray beam are discussed. Methods for immobilizing enzymes on the electrodes, while maintaining full redox control are highlighted. A case study of combined XAS and electrochemistry applied to a [NiFe] hydrogenase is presented. By entrapping the [NiFe] hydrogenase in a redox polymer, relatively high protein concentrations can be achieved on the electrode surface, while maintaining redox control. Overall, it is demonstrated that the experiments are feasible, but require precise redox control over the majority of the absorber atoms and careful controls to discriminate between electrochemically-driven changes and beam damage. Opportunities for future applications are discussed.

Surface Boron Modulation on Cobalt Oxide Nanocrystals for Electrochemical Oxygen Evolution Reaction.

Herein, we show that coupling boron with cobalt oxide tunes its structure and significantly boost its electrocatalytic performance for the oxygen evolution reaction (OER). Through a simple precipitation and thermal treatment process, a series of Co-B oxides with tunable morphologies and textural parameters were prepared. Detailed structural analysis supported first the formation of an disordered and partially amorphous material with nanosized Co3 BO5 and/or Co2 B2 O6 being present on the local atomic scale. The boron modulation resulted in a superior OER reactivity by delivering a large current and an overpotential of 338 mV to reach a current density of 10 mA cm-2 in 1 M KOH electrolyte. Identical location transmission electron microscopy and in situ electrochemical Raman spectroscopy studies revealed alteration and surface re-construction of materials, and formation of CoO2 and (oxy)hydroxide intermediate, which were found to be highly dependent on crystallinity of the samples.

The Nonphysiological Reductant Sodium Dithionite and [FeFe] Hydrogenase: Influence on the Enzyme Mechanism.

[FeFe] hydrogenases are highly active enzymes for interconverting protons and electrons with hydrogen (H2). Their active site H-cluster is formed of a canonical [4Fe-4S] cluster ([4Fe-4S]H) covalently attached to a unique [2Fe] subcluster ([2Fe]H), where both sites are redox active. Heterolytic splitting and formation of H2 takes place at [2Fe]H, while [4Fe-4S]H stores electrons. The detailed catalytic mechanism of these enzymes is under intense investigation, with two dominant models existing in the literature. In one model, an alternative form of the active oxidized state Hox, named HoxH, which forms at low pH in the presence of the nonphysiological reductant sodium dithionite (NaDT), is believed to play a crucial role. HoxH was previously suggested to have a protonated [4Fe-4S]H. Here, we show that HoxH forms by simple addition of sodium sulfite (Na2SO3, the dominant oxidation product of NaDT) at low pH. The low pH requirement indicates that sulfur dioxide (SO2) is the species involved. Spectroscopy supports binding at or near [4Fe-4S]H, causing its redox potential to increase by ∼60 mV. This potential shift detunes the redox potentials of the subclusters of the H-cluster, lowering activity, as shown in protein film electrochemistry (PFE). Together, these results indicate that HoxH and its one-electron reduced counterpart Hred'H are artifacts of using a nonphysiological reductant, and not crucial catalytic intermediates. We propose renaming these states as the "dithionite (DT) inhibited" states Hox-DTi and Hred-DTi. The broader potential implications of using a nonphysiological reductant in spectroscopic and mechanistic studies of enzymes are highlighted.

Surface-Promoted Evolution of Ru-bda Coordination Oligomers Boosts the Efficiency of Water Oxidation Molecular Anodes.

A new Ru oligomer of formula {[RuII(bda-κ-N2O2)(4,4'-bpy)]10(4,4'-bpy)}, 10 (bda is [2,2'-bipyridine]-6,6'-dicarboxylate and 4,4'-bpy is 4,4'-bipyridine), was synthesized and thoroughly characterized with spectroscopic, X-ray, and electrochemical techniques. This oligomer exhibits strong affinity for graphitic materials through CH-π interactions and thus easily anchors on multiwalled carbon nanotubes (CNT), generating the molecular hybrid material 10@CNT. The latter acts as a water oxidation catalyst and converts to a new species, 10'(H2O)2@CNT, during the electrochemical oxygen evolution process involving solvation and ligand reorganization facilitated by the interactions of molecular Ru catalyst and the surface. This heterogeneous system has been shown to be a powerful and robust molecular hybrid anode for electrocatalytic water oxidation into molecular oxygen, achieving current densities in the range of 200 mA/cm2 at pH 7 under an applied potential of 1.45 V vs NHE. The remarkable long-term stability of this hybrid material during turnover is rationalized based on the supramolecular interaction of the catalyst with the graphitic surface.

Spectroscopic and biochemical insight into an electron-bifurcating [FeFe] hydrogenase.

The heterotrimeric electron-bifurcating [FeFe] hydrogenase (HydABC) from Thermotoga maritima (Tm) couples the endergonic reduction of protons (H+) by dihydronicotinamide adenine dinucleotide (NADH) (∆G0 ≈ 18 kJ mol-1) to the exergonic reduction of H+ by reduced ferredoxin (Fdred) (∆G0 ≈ - 16 kJ mol-1). The specific mechanism by which HydABC functions is not understood. In the current study, we describe the biochemical and spectroscopic characterization of TmHydABC recombinantly produced in Escherichia coli and artificially maturated with a synthetic diiron cofactor. We found that TmHydABC catalyzed the hydrogen (H2)-dependent reduction of nicotinamide adenine dinucleotide (NAD+) in the presence of oxidized ferredoxin (Fdox) at a rate of ≈17 µmol NADH min-1 mg-1. Our data suggest that only one flavin is present in the enzyme and is not likely to be the site of electron bifurcation. FTIR and EPR spectroscopy, as well as FTIR spectroelectrochemistry, demonstrated that the active site for H2 conversion, the H-cluster, in TmHydABC behaves essentially the same as in prototypical [FeFe] hydrogenases, and is most likely also not the site of electron bifurcation. The implications of these results are discussed with respect to the current hypotheses on the electron bifurcation mechanism of [FeFe] hydrogenases. Overall, the results provide insight into the electron-bifurcating mechanism and present a well-defined system for further investigations of this fascinating class of [FeFe] hydrogenases.

Ruthenium 4d-to-2p X-ray Emission Spectroscopy: A Simultaneous Probe of the Metal and the Bound Ligands.

Ruthenium 4d-to-2p X-ray emission spectroscopy (XES) was systematically explored for a series of Ru2+ and Ru3+ species. Complementary density functional theory calculations were utilized to allow for a detailed assignment of the experimental spectra. The studied complexes have a range of different coordination spheres, which allows the influence of the ligand donor/acceptor properties on the spectra to be assessed. Similarly, the contributions of the site symmetry and the oxidation state of the metal were analyzed. Because the 4d-to-2p emission lines are dipole-allowed, the spectral features are intense. Furthermore, in contrast with K- or L-edge X-ray absorption of 4d transition metals, which probe the unoccupied levels, the observed 4p-to-2p XES arises from electrons in filled-ligand- and filled-metal-based orbitals, thus providing simultaneous access to the ligand and metal contributions to bonding. As such, 4d-to-2p XES should be a promising tool for the study of a wide range of 4d transition-metal compounds.

His-Ligation to the [4Fe-4S] Subcluster Tunes the Catalytic Bias of [FeFe] Hydrogenase.

[FeFe] hydrogenases interconvert H2 into protons and electrons reversibly and efficiently. The active site H-cluster is composed of two sites: a unique [2Fe] subcluster ([2Fe]H) covalently linked via cysteine to a canonical [4Fe-4S] cluster ([4Fe-4S]H). Both sites are redox active and electron transfer is proton-coupled, such that the potential of the H-cluster lies very close to the H2 thermodynamic potential, which confers the enzyme with the ability to operate quickly in both directions without energy losses. Here, one of the cysteines coordinating [4Fe-4S]H (Cys362) in the [FeFe] hydrogenase from the green algae Chlamydomonas reinhardtii ( CrHydA1) was exchanged with histidine and the resulting C362H variant was shown to contain a [4Fe-4S] cluster with a more positive redox potential than the wild-type. The change in the [4Fe-4S] cluster potential resulted in a shift of the catalytic bias, diminishing the H2 production activity but giving significantly higher H2 oxidation activity, albeit with a 200 mV overpotential requirement. These results highlight the importance of the [4Fe-4S] cluster as an electron injection site, modulating the redox potential and the catalytic properties of the H-cluster.

Unique Spectroscopic Properties of the H-Cluster in a Putative Sensory [FeFe] Hydrogenase.

Sensory type [FeFe] hydrogenases are predicted to play a role in transcriptional regulation by detecting the H2 level of the cellular environment. These hydrogenases contain the hydrogenase domain with distinct modifications in the active site pocket, followed by a Per-Arnt-Sim (PAS) domain. As yet, neither the physiological function nor the biochemical or spectroscopic properties of these enzymes have been explored. Here, we present the characterization of an artificially maturated, putative sensory [FeFe] hydrogenase from Thermotoga maritima (HydS). This enzyme shows lower hydrogen conversion activity than prototypical [FeFe] hydrogenases and a reduced inhibition by CO. Using FTIR spectroelectrochemistry and EPR spectroscopy, three redox states of the active site were identified. The spectroscopic signatures of the most oxidized state closely resemble those of the Hox state from the prototypical [FeFe] hydrogenases, while the FTIR spectra of both singly and doubly reduced states show large differences. The FTIR bands of both the reduced states are strongly red-shifted relative to the Hox state, indicating reduction at the diiron site, but with retention of the bridging CO ligand. The unique functional and spectroscopic features of HydS are discussed with regard to the possible role of altered amino acid residues influencing the electronic properties of the H-cluster.

Sulfide Protects [FeFe] Hydrogenases From O2.

[FeFe] hydrogenases catalyze proton reduction and hydrogen oxidation with high rates and efficiency under physiological conditions, but are highly oxygen sensitive. The [FeFe] hydrogenase from Desulfovibrio desulfuricans ( DdHydAB) can be purified under air in an oxygen stable inactive state Hoxair. The formation of the Hoxair state in vitro allows the handling of hydrogenases in air, making their implementation in biotechnological applications more feasible. Here, we report a simple and robust protocol for the formation of the Hoxair state in DdHydAB and the [FeFe] hydrogenase from Chlamydomonas reinhardtii, which is based on high potential inactivation in the presence of sulfide.

The structurally unique photosynthetic Chlorella variabilis NC64A hydrogenase does not interact with plant-type ferredoxins.

Hydrogenases from green algae are linked to the photosynthetic electron transfer chain via the plant-type ferredoxin PetF. In this work the [FeFe]-hydrogenase from the Trebouxiophycean alga Chlorella variabilis NC64A (CvHydA1), which in contrast to other green algal hydrogenases contains additional FeS-cluster binding domains, was purified and specific enzyme activities for both hydrogen (H2) production and H2 oxidation were determined. Interestingly, although C. variabilis NC64A, like many Chlorophycean algal strains, exhibited light-dependent H2 production activity upon sulfur deprivation, CvHydA1 did not interact in vitro with several plant-type [2Fe-2S]-ferredoxins, but only with a bacterial2[4Fe4S]-ferredoxin. In an electrochemical characterization, the enzyme exhibited features typical of bacterial [FeFe]-hydrogenases (e.g. minor anaerobic oxidative inactivation), as well as of algal enzymes (very high oxygen sensitivity).

Intercluster Redox Coupling Influences Protonation at the H-cluster in [FeFe] Hydrogenases.

[FeFe] hydrogenases catalyze proton reduction and hydrogen oxidation displaying high rates at low overpotential. Their active site is a complex cofactor consisting of a unique [2Fe] subcluster ([2Fe]H) covalently bound to a canonical [4Fe-4S] cluster ([4Fe-4S]H). The [FeFe] hydrogenase from Desulfovibrio desulfuricans is exceptionally active and bidirectional. This enzyme features two accessory [4Fe-4S]F clusters for exchanging electrons with the protein surface. A thorough understanding of the mechanism of this efficient enzyme will facilitate the development of synthetic molecular catalysts for hydrogen conversion. Here, it is demonstrated that the accessory clusters influence the catalytic properties of the enzyme through a strong redox interaction between the proximal [4Fe-4S]F cluster and the [4Fe-4S]H subcluster of the H-cluster. This interaction enhances proton-coupled electronic rearrangement within the H-cluster increasing the apparent pKa of its one electron reduced state. This may help to sustain H2 production at high pH values. These results may apply to all [FeFe] hydrogenases containing accessory clusters.

Proton Coupled Electronic Rearrangement within the H-Cluster as an Essential Step in the Catalytic Cycle of [FeFe] Hydrogenases.

The active site of [FeFe] hydrogenases, the H-cluster, consists of a [4Fe-4S] cluster connected via a bridging cysteine to a [2Fe] complex carrying CO and CN- ligands as well as a bridging aza-dithiolate ligand (ADT) of which the amine moiety serves as a proton shuttle between the protein and the H-cluster. During the catalytic cycle, the two subclusters change oxidation states: [4Fe-4S]H2+ ⇔ [4Fe-4S]H+ and [Fe(I)Fe(II)]H ⇔ [Fe(I)Fe(I)]H thereby enabling the storage of the two electrons needed for the catalyzed reaction 2H+ + 2e- ⇄ H2. Using FTIR spectro-electrochemistry on the [FeFe] hydrogenase from Chlamydomonas reinhardtii (CrHydA1) at different pH values, we resolve the redox and protonation events in the catalytic cycle and determine their intrinsic thermodynamic parameters. We show that the singly reduced state Hred of the H-cluster actually consists of two species: Hred = [4Fe-4S]H+ - [Fe(I)Fe(II)]H and HredH+ = [4Fe-4S]H2+ - [Fe(I)Fe(I)]H (H+) related by proton coupled electronic rearrangement. The two redox events in the catalytic cycle occur on the [4Fe-4S]H subcluster at similar midpoint-potentials (-375 vs -418 mV); the protonation event (Hred/HredH+) has a pKa ≈ 7.2.

Interplay between CN- Ligands and the Secondary Coordination Sphere of the H-Cluster in [FeFe]-Hydrogenases.

The catalytic cofactor of [FeFe]-hydrogenses (H-cluster) is composed of a generic cubane [4Fe-4S]-cluster (4FeH) linked to a binuclear iron-sulfur cluster (2FeH) that has an open coordination site at which the reversible conversion of protons to molecular hydrogen occurs. The (2FeH) subsite features a diatomic coordination sphere composed of three CO and two CN- ligands affecting its redox properties and providing excellent probes for FTIR spectroscopy. The CO stretch vibrations are very sensitive to the redox changes within the H-cluster occurring during the catalytic cycle, whereas the CN- signals seem to be relatively inert to these effects. This could be due to the more structural role of the CN- ligands tightly anchoring the (2FeH) unit to the protein environment through hydrogen bonding. In this work we explore the effects of structural changes within the secondary ligand sphere affecting the CN- ligands on FTIR spectroscopy and catalysis. By comparing the FTIR spectra of wild-type enzyme and two mutagenesis variants, we are able to assign the IR signals of the individual CN- ligands of the (2FeH) site for different redox states of the H-cluster. Moreover, protein film electrochemistry reveals that targeted manipulation of the secondary coordination sphere of the proximal CN- ligand (i.e., closest to the (4FeH) site) can affect the catalytic bias. These findings highlight the importance of the protein environment for re-adjusting the catalytic features of the H-cluster in individual enzymes and provide valuable information for the design of artificial hydrogenase mimics.

Mechanism of protection of catalysts supported in redox hydrogel films.

The use of synthetic inorganic complexes as supported catalysts is a key route in energy production and in industrial synthesis. However, their intrinsic oxygen sensitivity is sometimes an issue. Some of us have recently demonstrated that hydrogenases, the fragile but very efficient biological catalysts of H2 oxidation, can be protected from O2 damage upon integration into a film of a specifically designed redox polymer. Catalytic oxidation of H2 produces electrons which reduce oxygen near the film/solution interface, thus providing a self-activated protection from oxygen [Plumeré et al., Nat Chem. 2014, 6, 822-827]. Here, we rationalize this protection mechanism by examining the time-dependent distribution of species in the hydrogenase/polymer film, using measured or estimated values of all relevant parameters and the numerical and analytical solutions of a realistic reaction-diffusion scheme. Our investigation sets the stage for optimizing the design of hydrogenase-polymer films, and for expanding this strategy to other fragile catalysts.

Direct comparison of the performance of a bio-inspired synthetic nickel catalyst and a [NiFe]-hydrogenase, both covalently attached to electrodes.

The active site of hydrogenases has been a source of inspiration for the development of molecular catalysts. However, direct comparisons between molecular catalysts and enzymes have not been possible because different techniques are used to evaluate both types of catalysts, minimizing our ability to determine how far we have come in mimicking the enzymatic performance. The catalytic properties of the [Ni(P(Cy) 2 N(Gly) 2 )2 ](2+) complex with the [NiFe]-hydrogenase from Desulfovibrio vulgaris immobilized on a functionalized electrode were compared under identical conditions. At pH 7, the enzyme shows higher activity and lower overpotential with better stability, while at low pH, the molecular catalyst outperforms the enzyme in all respects. This is the first direct comparison of enzymes and molecular complexes, enabling a unique understanding of the benefits and detriments of both systems, and advancing our understanding of the utilization of these bio-inspired complexes in fuel cells.

A redox hydrogel protects the O2 -sensitive [FeFe]-hydrogenase from Chlamydomonas reinhardtii from oxidative damage.

The integration of sensitive catalysts in redox matrices opens up the possibility for their protection from deactivating molecules such as O2 . [FeFe]-hydrogenases are enzymes catalyzing H2 oxidation/production which are irreversibly deactivated by O2 . Therefore, their use under aerobic conditions has never been achieved. Integration of such hydrogenases in viologen-modified hydrogel films allows the enzyme to maintain catalytic current for H2 oxidation in the presence of O2 , demonstrating a protection mechanism independent of reactivation processes. Within the hydrogel, electrons from the hydrogenase-catalyzed H2 oxidation are shuttled to the hydrogel-solution interface for O2 reduction. Hence, the harmful O2 molecules do not reach the hydrogenase. We illustrate the potential applications of this protection concept with a biofuel cell under H2 /O2 mixed feed.

[NiFe] hydrogenases: a common active site for hydrogen metabolism under diverse conditions.

Hydrogenase proteins catalyze the reversible conversion of molecular hydrogen to protons and electrons. The most abundant hydrogenases contain a [NiFe] active site; these proteins are generally biased towards hydrogen oxidation activity and are reversibly inhibited by oxygen. However, there are [NiFe] hydrogenase that exhibit unique properties, including aerobic hydrogen oxidation and preferential hydrogen production activity; these proteins are highly relevant in the context of biotechnological devices. This review describes four classes of these "nonstandard" [NiFe] hydrogenases and discusses the electrochemical, spectroscopic, and structural studies that have been used to understand the mechanisms behind this exceptional behavior. A revised classification protocol is suggested in the conclusions, particularly with respect to the term "oxygen-tolerance". This article is part of a special issue entitled: metals in bioenergetics and biomimetics systems.