Structural Determinants of the Catalytic Nia-L Intermediate of [NiFe]-Hydrogenase.

[NiFe]-hydrogenases catalyze the reversible cleavage of H2 into two protons and two electrons at the inorganic heterobimetallic NiFe center of the enzyme. Their catalytic cycle involves at least four intermediates, some of which are still under debate. While the core reaction, including H2/H- binding, takes place at the inorganic cofactor, a major challenge lies in identifying those amino acid residues that contribute to the reactivity and how they stabilize (short-lived) intermediate states. Using cryogenic infrared and electron paramagnetic resonance spectroscopy on the regulatory [NiFe]-hydrogenase from Cupriavidus necator, a model enzyme for the analysis of catalytic intermediates, we deciphered the structural basis of the hitherto elusive Nia-L intermediates. We unveiled the protonation states of a proton-accepting glutamate and a Ni-bound cysteine residue in the Nia-L1, Nia-L2, and the hydride-binding Nia-C intermediates as well as previously unknown conformational changes of amino acid residues in proximity of the bimetallic active site. As such, this study unravels the complexity of the Nia-L intermediate and reveals the importance of the protein scaffold in fine-tuning proton and electron dynamics in [NiFe]-hydrogenase.

Substrate-Gated Transformation of a Pre-Catalyst into an Iron-Hydride Intermediate [(NO)2(CO)Fe(µ-H)Fe(CO)(NO)2]- for Catalytic Dehydrogenation of Dimethylamine Borane.

Continued efforts are made on the development of earth-abundant metal catalysts for dehydrogenation/hydrolysis of amine boranes. In this study, complex [K-18-crown-6-ether][(NO)2Fe(µ-MePyr)(µ-CO)Fe(NO)2] (3-K-crown, MePyr = 3-methylpyrazolate) was explored as a pre-catalyst for the dehydrogenation of dimethylamine borane (DMAB). Upon evolution of H2(g) from DMAB triggered by 3-K-crown, parallel conversion of 3-K-crown into [(NO)2Fe(N,N'-MePyrBH2NMe2)]- (5) and an iron-hydride intermediate [(NO)2(CO)Fe(µ-H)Fe(CO)(NO)2]- (A) was evidenced by X-ray diffraction/nuclear magnetic resonance/infrared/nuclear resonance vibrational spectroscopy experiments and supported by density functional theory calculations. Subsequent transformation of A into complex [(NO)2Fe(µ-CO)2Fe(NO)2]- (6) is synchronized with the deactivated generation of H2(g). Through reaction of complex [Na-18-crown-6-ether][(NO)2Fe(η2-BH4)] (4-Na-crown) with CO(g) as an alternative synthetic route, isolated intermediate [Na-18-crown-6-ether][(NO)2(CO)Fe(µ-H)Fe(CO)(NO)2] (A-Na-crown) featuring catalytic reactivity toward dehydrogenation of DMAB supports a substrate-gated transformation of a pre-catalyst [(NO)2Fe(µ-MePyr)(µ-CO)Fe(NO)2]- (3) into the iron-hydride species A as an intermediate during the generation of H2(g).

Enhancement of Peroxidase Activity in Artificial Mimochrome†VI Catalysts through Rational Design.

Rational design provides an attractive strategy to tune and control the reactivity of bioinspired catalysts. Although there has been considerable progress in the design of heme oxidase mimetics with active-site environments of ever-growing complexity and catalytic efficiency, their stability during turnover is still an open challenge. Herein, we show that the simple incorporation of two 2-aminoisobutyric acids into an artificial peptide-based peroxidase results in a new catalyst (FeIII -MC6*a) with higher resistance against oxidative damage and higher catalytic efficiency. The turnover number of this catalyst is twice as high as that of its predecessor. These results point out the protective role exerted by the peptide matrix and pave the way to the synthesis of robust bioinspired catalysts.

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.

From enzyme maturation to synthetic chemistry: the case of hydrogenases.

Water splitting into oxygen and hydrogen is one of the most attractive strategies for storing solar energy and electricity. Because the processes at work are multielectronic, there is a crucial need for efficient and stable catalysts, which in addition have to be cheap for future industrial developments (electrolyzers, photoelectrochemicals, and fuel cells). Specifically for the water/hydrogen interconversion, Nature is an exquisite source of inspiration since this chemistry contributes to the bioenergetic metabolism of a number of living organisms via the activity of fascinating metalloenzymes, the hydrogenases. In this Account, we first briefly describe the structure of the unique dinuclear organometallic active sites of the two classes of hydrogenases as well as the complex protein machineries involved in their biosynthesis, their so-called maturation processes. This knowledge allows for the development of a fruitful bioinspired chemistry approach, which has already led to a number of interesting and original catalysts mimicking the natural active sites. More specifically, we describe our own attempts to prepare artificial hydrogenases. This can be achieved via the standard bioinspired approach using the combination of a synthetic bioinspired catalyst and a polypeptide scaffold. Such hybrid complexes provide the opportunity to optimize the system by manipulating both the catalyst through chemical synthesis and the protein component through mutagenesis. We also raise the possibility to reach such artificial systems via an original strategy based on mimicking the enzyme maturation pathways. This is illustrated in this Account by two examples developed in our laboratory. First, we show how the preparation of a lysozyme-{Mn(I)(CO)3} hybrid and its clean reaction with a nickel complex led us to generate a new class of binuclear Ni-Mn H2-evolving catalysts mimicking the active site of [NiFe]-hydrogenases. Then we describe how we were able to rationally design and prepare a hybrid system, displaying remarkable structural similarities to an [FeFe]-hydrogenase, and we show here for the first time that it is catalytically active for proton reduction. This system is based on the combination of HydF, a protein involved in the maturation of [FeFe]-hydrogenase (HydA), and a close mimic of the active site of this class of enzymes. Moreover, the synthetic [Fe2(adt)(CO)4(CN)2](2-) (adt(2-)= aza-propanedithiol) mimic, alone or within a HydF hybrid system, was shown to be able to maturate and activate a form of HydA itself lacking its diiron active site. We discuss the exciting perspectives this "synthetic maturation" opens regarding the "invention" of novel hydrogenases by the chemists.

An artificial heme-enzyme with enhanced catalytic activity: evolution, functional screening and structural characterization.

Synthetic proteins represent useful tools for reproducing metalloprotein functions in minimal, well-defined scaffolds. Herein, we describe the rational refinement of function into heme-protein models from the Mimochrome family. Originally designed to mimic the bis-His cytochrome b, the Mimochrome structure was modified to introduce a peroxidase-like activity, by creating a distal cavity on the heme. The success with the first asymmetric system, Mimochrome VI (MC6), gave the opportunity to explore further modifications in order to improve the catalytic activity. Starting from ferric MC6, single amino acid substitutions were introduced in the peptide chains to obtain four compounds, which were screened for peroxidase activity. The detailed structural and functional analysis of the best analogue, Fe(III)-E(2)L(TD)-MC6, indicates that an arginine residue in proximity to the heme-distal site could assist with catalysis by favoring the formation of the intermediate "compound I", thus mimicking R(38) in HRP. This result highlights the potential of using small scaffolds for exploring the main factors that tune the heme-protein activity, and for programming new desired functions.