Hydrogenases are the most active molecular catalysts for hydrogen production and uptake on earth 1,2 and are thus extensively studied with respect to their technological exploitation as noble metal substitutes in (photo)electrolysers and fuel cells [3][4][5] . In [FeFe]-hydrogenases catalysis takes place at a unique diiron center (the [2Fe] subsite) featuring a bridging dithiolate ligand, as well as three CO and two CN − ligands (Figure 1) 6,7 . Through a complex and as yet poorly understood multienzymatic biosynthetic process, this [2Fe] subsite is first assembled onto a maturation enzyme, HydF. From there, it is delivered to the apo-hydrogenase for activation 8 . Synthetic chemistry has allowed the preparation of remarkably close mimics of that subsite 1 but failed to reproduce the natural enzymatic activities so far. Here we show that three such synthetic mimics (with different bridging dithiolate ligands) can be loaded onto HydF and then transferred to apoHydA1, one of the hydrogenases of Chlamydomonas reinhardtii. Remarkably, full activation of HydA1 was achieved exclusively using the HydF hybrid protein containing the mimic with an azadithiolate bridge, confirming the presence of this ligand in the active site of 10 . This is the first example of controlled metalloenzyme activation using the combination of a specific protein scaffold and active site synthetic analogues. This simple methodology provides both new mechanistic and structural insight into hydrogenase maturation and a unique tool for producing recombinant wild-type and variant [FeFe] cluster 17 and named "HydF" in the following, with a 10-fold molar excess of complex 1, 2 or 3, led to new hybrid species x-HydF (x = 1, 2 or 3 respectively), that could be isolated in pure form and characterized. In all cases, iron quantification indeed showed an increase from 3.9 ± 0.4 to 5.6 ± 0.4 iron atoms per protein and the UV-visible spectrum of these hybrids displayed features consistent with a ~1:1 ratio of the synthetic complexes and the HydF protein ( Figure S1a-c).FTIR spectroscopy is a convenient method for characterizing metalloproteins such as hydrogenases containing CO and CN − ligands 18 . Thus, further evidence for the incorporation of synthetic complexes in HydF was obtained from their FTIR spectra which contained CN − stretching bands between 2000 and 2100 cm −1 and four partly overlapping CO-stretching bands in the 1800-2000 cm −1 range ( Figure 2B and Table S1). The highenergy bands underwent a 40 cm −1 shift upon 13 C-labeling of the CN − ligands ( Figure S2). Interestingly, the width of the FTIR bands is still identical to those of the unbound complexes ( Figure 2A) but their positions show strong similarities with those of CaHydF ( Figure 2B and The arrangement in which the synthetic complexes are bound to HydF and its [4Fe-4S] cluster is not evident from the FTIR spectra. In particular FTIR spectroscopy does not allow to definitively distinguish between terminal and bridging cyanide ligands (see below and supplementary discussion) ...

Hydrogenases catalyze the formation of hydrogen. The cofactor ('H-cluster') of [FeFe]-hydrogenases consists of a [4Fe-4S] cluster bridged to a unique [2Fe] subcluster whose biosynthesis in vivo requires hydrogenase-specific maturases. Here we show that a chemical mimic of the [2Fe] subcluster can reconstitute apo-hydrogenase to full activity, independent of helper proteins. The assembled H-cluster is virtually indistinguishable from the native cofactor. This procedure will be a powerful tool for developing new artificial H₂-producing catalysts.

H2 turnover at the [FeFe]-hydrogenase cofactor (H-cluster) is assumed to follow a reversible heterolytic mechanism, first yielding a proton and a hydrido-species which again is double-oxidized to release another proton. Three of the four presumed catalytic intermediates (Hox, Hred/Hred and Hsred) were characterized, using various spectroscopic techniques. However, in catalytically active enzyme, the state containing the hydrido-species, which is eponymous for the proposed heterolytic mechanism, has yet only been speculated about. We use different strategies to trap and spectroscopically characterize this transient hydride state (Hhyd) for three wild-type [FeFe]-hydrogenases. Applying a novel set-up for real-time attenuated total-reflection Fourier-transform infrared spectroscopy, we monitor compositional changes in the state-specific infrared signatures of [FeFe]-hydrogenases, varying buffer pH and gas composition. We selectively enrich the equilibrium concentration of Hhyd, applying Le Chatelier’s principle by simultaneously increasing substrate and product concentrations (H2/H+). Site-directed manipulation, targeting either the proton-transfer pathway or the adt ligand, significantly enhances Hhyd accumulation independent of pH.

Crystal structures of semisynthetic [FeFe]-hydrogenases with variations in the [2Fe] cluster show little structural differences despite strong effects on activity.

The unmatched catalytic turnover rates of [FeFe]-hydrogenases require an exceptionally efficient proton-transfer (PT) pathway to shuttle protons as substrates or products between bulk water and catalytic center. For clostridial [FeFe]-hydrogenase CpI such a pathway has been proposed and analyzed, but mainly on a theoretical basis. Here, eleven enzyme variants of two different [FeFe]-hydrogenases (CpI and HydA1) with substitutions in the presumptive PT-pathway are examined kinetically, spectroscopically, and crystallographically to provide solid experimental proof for its role in hydrogen-turnover. Targeting key residues of the PT-pathway by site directed mutagenesis significantly alters the pH-activity profile of these variants and in presence of H2 their cofactor is trapped in an intermediate state indicative of precluded proton-transfer. Furthermore, crystal structures coherently explain the individual levels of residual activity, demonstrating e.g. how trapped H2O molecules rescue the interrupted PT-pathway. These features provide conclusive evidence that the targeted positions are indeed vital for catalytic proton-transfer.

A large fraction of proteins naturally exist as symmetrical homooligomers or homopolymers 1. The emergent structural and functional properties of such protein assemblies have inspired extensive efforts in biomolecular design 2-5. As synthesized by ribosomes, proteins are inherently asymmetric. Thus, they must acquire multiple surface patches that selectively associate to generate different symmetry elements needed to form higher-order architectures 1,6-a daunting task for protein design. Here we introduce an inorganic chemical approach to address this outstanding problem, whereby multiple modes of protein-protein interactions and symmetry are simultaneously achieved by selective, "one-pot" coordination of soft and hard metal ions. We show that a monomeric protein (protomer) appropriately modified with biologically inspired hydroxamate groups and Zn-binding motifs assembles through concurrent Fe 3+ and Zn 2+ coordination into discrete dodecameric and hexameric cages. Closely resembling natural polyhedral protein architectures 7,8 and unique among designed systems 9-13 , our artificial cages possess tightly packed shells devoid of large apertures, yet they can assemble and disassemble in response to diverse stimuli owing to their heterobimetallic construction on minimal interproteinbonding footprints. With stoichiometries ranging from [2 Fe:9 Zn:6 protomer] to [8 Fe:21 Zn:12 protomer], these protein cages represent some of the compositionally most complex protein assemblies-or inorganic coordination complexes-obtained by design. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

The precise electrochemical features of metal cofactors that convey the functions of redox enzymes are essentially determined by the specific interaction pattern between cofactor and enclosing protein environment. However, while biophysical techniques allow a detailed understanding of the features characterizing the cofactor itself, knowledge about the contribution of the protein part is much harder to obtain. [FeFe]-hydrogenases are an interesting class of enzymes that catalyze both, H2 oxidation and the reduction of protons to molecular hydrogen with significant efficiency. The active site of these proteins consists of an unusual prosthetic group (H-cluster) with six iron and six sulfur atoms. While H-cluster architecture and catalytic states during the different steps of H2 turnover have been thoroughly investigated during the last 20 years, possible functional contributions from the polypeptide framework were only assumed according to the level of conservancy and X-ray structure analyses. Due to the recent development of simpler and more efficient expression systems the role of single amino acids can now be experimentally investigated. This article summarizes, compares and categorizes the results of recent investigations based on site directed and random mutagenesis according to their informative value about structure function relationships in [FeFe]-hydrogenases. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems.

Protein film electrochemistry (PFE) has been used to study the assembly of the complex 6Fe active site of [FeFe]-hydrogenases (known as the H-cluster) from its precursors-the [4Fe-4S] domain that is already coordinated within the host, and the 2Fe domain that is presented as a synthetic water-soluble complex stabilized by an additional CO. Not only does PFE allow control of redox states via the electrode potential but also the immobilized state of the enzyme facilitates control of extremely low concentrations of the 2Fe complex. Results for two enzymes, CrHydA1 from Chlamydomonas reinhardtii and CpI from Clostridium pasteurianum, are very similar, despite large differences in size and structure. Assembly begins with very tight binding of the 34-valence electron 2Fe complex to the apo-[4Fe-4S] enzyme, well before the rate-determining step. The precursor is trapped under highly reducing conditions (<-0.5 V vs SHE) that prevent fusion of the [4Fe-4S] and 2Fe domains (via cysteine-S) since the immediate product would be too electron-rich. Relaxing this condition allows conversion to the active H-cluster. The intramolecular steps are relevant to the final stage of biological H-cluster maturation.