Souvik Roy

Artificial hydrogenases: biohybrid and supramolecular systems for catalytic hydrogen production or uptake

There is an urgent need for cheap, abundant and efficient catalysts as an alternative to platinum for hydrogen production and oxidation in (photo)electrolyzers and fuel cells. Hydrogenases are attractive solutions. These enzymes use exclusively nickel and iron in their active sites and function with high catalytic rates at the thermodynamic equilibrium. As an alternative, a number of biomimetic and bioinspired catalysts for H2 production and/or uptake, based on Ni, Fe and Co, have been developed and shown to display encouraging performances. In this review we discuss specifically recent approaches aiming at incorporating these compounds within oligomeric and polymeric hosts. The latter are most often biological compounds (peptides, proteins, polysaccharides, etc.) but we also discuss non-biological scaffolds (synthetic polymers, Metal-organic-Frameworks, etc.) which can provide the appropriate environment to tune the activity and stability of the synthetic catalysts. These supramolecular catalytic systems thus define a class of original compounds so-called artificial hydrogenases.

A Systematic Comparative Study of Hydrogen-Evolving Molecular Catalysts in Aqueous Solutions

We describe here a systematic, reliable, and fast screening method that allows the comparison of H2-forming catalysts that work under aqueous conditions with two readily prepared chemical reductants and two commonly used photosensitizers. This method uses a Clark-type microsensor for H2 detection and complements previous methods based on rotating disk electrode measurements. The efficiencies of a series of H2 -producing catalysts based on Co, Ni, Fe, and Pt were investigated in aqueous solutions under thermal conditions with europium(II) reductants and under photochemical conditions in the presence of two different photosensitizers {[Ru(bipy)3]Cl2(bipy=2,2-bipyridine) and eosin-Y} and sacrificial electron donors (ascorbate and triethanolamine, respectively). The majority of catalysts tested were active only under specific conditions. However, our results also demonstrate the impressive versatility of a group of Co catalysts, which were able to produce H2 under different reducing conditions and at various pH values. In particular, a cobaloxime, [Co(dmgH)2(H2O)2] (dmgH2 =dimethylglyoxime), and a cobalt tetraazamacrocyclic complex, {Co(CR)Cl2}(+) [CR=2,12-dimethyl-3,7,11,17-tetraazabicylo(11.3.1)heptadeca-1(17),2,11,13,15-pentaene], displayed excellent catalytic rates under the studied conditions, and the best rates were observed under thermal conditions.

Spectroscopic Characterization of the Bridging Amine in the Active Site of [FeFe] Hydrogenase Using Isotopologues of the H-Cluster

The active site of [FeFe] hydrogenase contains a catalytic binuclear iron subsite coordinated by CN(-) and CO ligands as well as a unique azadithiolate (adt(2-)) bridging ligand. It has been established that this binuclear cofactor is synthesized and assembled by three maturation proteins HydE, -F, and -G. By means of in vitro maturation in the presence of (15)N- and (13)C-labeled tyrosine it has been shown that the CN(-) and CO ligands originate from tyrosine. The source of the bridging adt(2-) ligand, however, remains unknown. In order to identify the nitrogen of the bridging amine using HYSCORE spectroscopy and distinguish its spectroscopic signature from that of the CN(-) nitrogens, we studied three isotope-labeled variants of the H-cluster ((15)N-adt(2-)/C(14)N(-), (15)N-adt(2-)/C(15)N(-), and (14)N-adt(2-)/C(15)N(-)) and extracted accurate values of the hyperfine and quadrupole couplings of both CN(-) and adt(2-) nitrogens. This will allow an evaluation of isotopologues of the H-cluster generated by in vitro bioassembly in the presence of various (15)N-labeled potential precursors as possible sources of the bridging ligand.

Biomimetic peptide-based models of [FeFe]-hydrogenases: utilization of phosphine-containing peptides

Two synthetic strategies for incorporating diiron analogues of [FeFe]-hydrogenases into short peptides via phosphine functional groups are described. First, utilizing the amine side chain of lysine as an anchor, phosphine carboxylic acids can be coupled via amide formation to resin-bound peptides. Second, artificial, phosphine-containing amino acids can be directly incorporated into peptides via solution phase peptide synthesis. The second approach is demonstrated using three amino acids each with a different phosphine substituent (diphenyl, diisopropyl, and diethyl phosphine). In total, five distinct monophosphine-substituted, diiron model complexes were prepared by reaction of the phosphine-peptides with diiron hexacarbonyl precursors, either (μ-pdt)Fe2(CO)6 or (μ-bdt)Fe2(CO)6 (pdt = propane-1,3-dithiolate, bdt = benzene-1,2-dithiolate). Formation of the complexes was confirmed by UV/Vis, FTIR and (31)P NMR spectroscopy. Electrocatalysis by these complexes is reported in the presence of acetic acid in mixed aqueous-organic solutions. Addition of water results in enhancement of the catalytic rates.

Chemical assembly of multiple metal cofactors: The heterologously expressed multidomain [FeFe]-hydrogenase from Megasphaera elsdenii

[FeFe]-hydrogenases are unique and fascinating enzymes catalyzing the reversible reduction of protons into hydrogen. These metalloenzymes display extremely large catalytic reaction rates at very low overpotential values and are, therefore, studied as potential catalysts for bioelectrodes of electrolyzers and fuel cells. Since they contain multiple metal cofactors whose biosynthesis depends on complex protein machineries, their preparation is difficult. As a consequence still few have been purified to homogeneity allowing spectroscopic and structural characterization. As part of a program aiming at getting easy access to new hydrogenases we report here a methodology based on a purely chemical assembly of their metal cofactors. This methodology is applied to the preparation and characterization of the hydrogenase from the fermentative anaerobic rumen bacterium Megasphaera elsdenii, which has only been incompletely characterized in the past.

Sequential oxidations of thiolates and the cobalt metallocenter in a synthetic metallopeptide: Implications for the biosynthesis of nitrile hydratase

Cobalt nitrile hydratases (Co-NHase) contain a catalytic cobalt(III) ion coordinated in an N2S3 first coordination sphere composed of two amidate nitrogens and three cysteine-derived sulfur donors: a thiolate (-SR), a sulfenate (-S(R)O(-)), and a sulfinate (-S(R)O2(-)). The sequence of biosynthetic reactions that leads to the post-translational oxidations of the metal and the sulfur ligands is unknown, but the process is believed to be initiated directly by oxygen. Herein we utilize cobalt bound in an N2S2 first coordination sphere by a seven amino acid peptide known as SODA (ACDLPCG) to model this oxidation process. Upon exposure to oxygen, Co-SODA is oxidized in two steps. In the first fast step (seconds), magnetic susceptibility measurements demonstrated that the metallocenter remains paramagnetic, that is, Co(2+), and sulfur K-edge X-ray absorption spectroscopy (XAS) is used to show that one of the thiolates is oxidized to sulfinate. In a second process on a longer time scale (hours), magnetic susceptibility measurements and Co K-edge XAS show that the metal is oxidized to Co(3+). Unlike other model complexes, additional slow oxidation of the second thiolate in Co-SODA is not observed, and a catalytically active complex is never formed. The likely reason is the absence of the axial thiolate ligand. In essence, the reactivity of Co-SODA can be described as between previously described models which either quickly convert to final product or are stable in air, and it offers a first glimpse into a possible oxidation pathway for nitrile hydratase biosynthesis.

Structural and functional characterization of the hydrogenase-maturation HydF protein

[FeFe] hydrogenase (HydA) catalyzes interconversion between 2H+ and H2 at an active site composed of a [4Fe-4S] cluster linked to a 2Fe subcluster that harbors CO, CN- and azapropanedithiolate (adt2-) ligands. HydE, HydG and HydF are the maturases specifically involved in the biosynthesis of the 2Fe subcluster. Using ligands synthesized by HydE and HydG, HydF assembles a di-iron precursor of the 2Fe subcluster and transfers it to HydA for maturation. Here we report the first X-ray structure of HydF with its [4Fe-4S] cluster. The cluster is chelated by three cysteines and an exchangeable glutamate, which allows the binding of synthetic mimics of the 2Fe subcluster. [Fe2(adt)(CO)4(CN)2]2- is proposed to be the true di-iron precursor because, when bound to HydF, it matures HydA and displays features in Fourier transform infrared (FTIR) spectra that are similar to those of the native HydF active intermediate. A new route toward the generation of artificial hydrogenases, as combinations of HydF and such biomimetic complexes, is proposed on the basis of the observed hydrogenase activity of chemically modified HydF.

A noble metal-free photocatalytic system based on a novel cobalt tetrapyridyl catalyst for hydrogen production in fully aqueous medium

The new cobalt tetrapyridyl complex 1(BF4)2 was characterized and assessed for hydrogen production in fully aqueous solution. Mechanistic information was gained thanks to a fast screening method, using chemical reductants and a Clark microelectrode. Optimal hydrogen production (443 TONs – 3 mL of H2) was achieved under visible light-driven conditions, in the presence of a noble metal-free photosensitizer, the water soluble porphyrin 2Cl4, and the ascorbate/tris-(2-carboxyethyl)phosphine (TCEP) sacrificial electron donor system.

Metalloenzymes: Cutting out the middleman

In vivo, hydrogenases require maturases for active site incorporation. However, in vitro, an active site model with limited catalytic activity could be incorporated into the apo form of [FeFe]-hydrogenase without the aid of maturases, generating enzyme with native activity.

Spectroscopic investigations of a semi-synthetic [FeFe] hydrogenase with propane di-selenol as bridging ligand in the binuclear subsite: comparison to the wild type and propane di-thiol variants

Abstract[FeFe] Hydrogenases catalyze the reversible conversion of H2 into electrons and protons. Their catalytic site, the H-cluster, contains a generic [4Fe–4S]H cluster coupled to a [2Fe]H subsite [Fe2(ADT)(CO)3(CN)2]2−, ADT = µ(SCH2)2NH. Heterologously expressed [FeFe] hydrogenases (apo-hydrogenase) lack the [2Fe]H unit, but this can be incorporated through artificial maturation with a synthetic precursor [Fe2(ADT)(CO)4(CN)2]2−. Maturation with a [2Fe] complex in which the essential ADT amine moiety has been replaced by CH2 (PDT = propane-dithiolate) results in a low activity enzyme with structural and spectroscopic properties similar to those of the native enzyme, but with simplified redox behavior. Here, we study the effect of sulfur-to-selenium (S-to-Se) substitution in the bridging PDT ligand incorporated in the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii using magnetic resonance (EPR, NMR), FTIR and spectroelectrochemistry. The resulting HydA1-PDSe enzyme shows the same redox behavior as the parent HydA1-PDT. In addition, a state is observed in which extraneous CO is bound to the open coordination site of the [2Fe]H unit. This state was previously observed only in the native enzyme HydA1-ADT and not in HydA1-PDT. The spectroscopic features and redox behavior of HydA1-PDSe, resulting from maturation with [Fe2(PDSe)(CO)4(CN)2]2−, are discussed in terms of spin and charge density shifts and provide interesting insight into the electronic structure of the H-cluster. We also studied the effect of S-to-Se substitution in the [4Fe–4S] subcluster. The reduced form of HydA1 containing only the [4Fe–4Se]H cluster shows a characteristic S = 7/2 spin state which converts back into the S = 1/2 spin state upon maturation with a [2Fe]–PDT/ADT complex.