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.

Over the last 15 years, a plethora of research has provided major insights into the structure and function of hydrogenase enzymes. This has led to the important development of chemical models that mimic the inorganic enzymatic co-factors, which in turn has further contributed to the understanding of the specific molecular features of these natural systems that facilitate such large and robust enzyme activities. More recently, efforts have been made to generate guest-host models and artificial hydrogenases, through the incorporation of transition metal-catalysts (guests) into various hosts. This adds a new layer of complexity to hydrogenase-like catalytic systems that allows for better tuning of their activity through manipulation of both the first (the guest) and the second (the host) coordination sphere. Herein we review the aforementioned advances achieved during the last 15 years, in the field of inorganic biomimetic hydrogenase chemistry. After a brief presentation of the enzymes themselves, as well as the early bioinspired catalysts, we review the more recent systems constructed as models for the hydrogenase enzymes, with a specific focus on the various strategies employed for incorporating of synthetic models into supramolecular frameworks and polypeptidic/protein scaffolds, and critically discuss the advantages of such an elaborate approach, with regard to the catalytic performances. evolving systems with enhanced activity, it has also recently provided a novel and exciting route for the direct and facile activation of native [FeFe] hydrogenases [10,11]. HydrogenasesCharacterization of certain living organisms, such as archaea, bacteria, cyanobacteria and algae, has led to the exciting discovery that hydrogen can be either produced or utilised as a source of low-potential electrons within living cells participating in a global H 2 cycle [12].Bacteria such as Ralstonia eutropha (a facultative chemolithoautotrophic organism) provide a good example of this as they are able to use hydrogen as their sole source of energy [13].Another example comes from micro-algaea such as Chlamydomonas reinhardtii, which under certain conditions is able to use sunlight to transiently drive the reverse reaction, i.e. extracting electrons from water and using them to reduce protons into hydrogen [14]. Finally, methanogens such as Methanobacterium thermoautotrophicum are able to exploit the reducing power of H 2 to produce CH 4 from CO 2 [15]. This chemical activity is made possible through the expression of fascinating metalloenzymes called hydrogenases [13,16,17]. There are two classes of hydrogenmetabolizing enzymes, the [NiFe]-and [FeFe]-hydrogenases, which catalyse these reactions without any overpotential [18] and at very high rates (one molecule of hydrogenase produces between 1500 to 20000 molecules of H 2 per second at pH 7 and 37 °C in water) [3,19,20]. A third class, [Fe]-hydrogenase or Hmd (Hydrogen-forming methylene-tetrahydromethanopterin dehydrogenase), is only found in archaea methanogens and requires the use of a...

Hydrogenases are redox enzymes that catalyze the conversion of protons and molecular hydrogen (H2). Based on the composition of the active site cofactor, the monometallic [Fe]hydrogenase is distinguished from the bimetallic [NiFe]-or [FeFe]-hydrogenase. The latter has been reported with particularly high turnover activities for both H2 release and H2 oxidation, notably at neutral pH, ambient temperatures, and negligible electric overpotential. Due to these properties, [FeFe]-hydrogenase represents the 'gold standard' in enzymatic hydrogen turnover.Understanding hydrogenase chemistry is crucial for the design of transition metal complexes that serve as potentially sustainable proton reduction or H2 oxidation catalysts, e.g. in electrolytic devices or fuel cells.Even 20 years after the crystal structures of [FeFe]-hydrogenase have been published, several aspects of biological hydrogen turnover are heatedly discussed. In this perspective, we give an overview on how the diversity of naturally occurring and artificially prepared, semi-synthetic [FeFe]-hydrogenases deepens our understanding of hydrogenase chemistry. In parallel, we cover recent results from biophysical techniques that go beyond the scope of conventional Xray diffraction, EPR, and FTIR spectroscopy. Taking into account both proton transfer and electron transfer as well as the notorious sensitivity of [FeFe]-hydrogenases towards carbon monoxide, the discussion further touches upon the molecular proceedings of biological hydrogen turnover.

In oxygenic photosynthesis, water is oxidized and dioxygen is produced at a Mn4Ca complex bound to the proteins of photosystem II (PSII). Valence and coordination changes in its catalytic S-state cycle are of great interest. In room-temperature (in situ) experiments, time-resolved energy-sampling X-ray emission spectroscopy of the Mn Kβ1,3 line after laser-flash excitation of PSII membrane particles was applied to characterize the redox transitions in the S-state cycle. The Kβ1,3 line energies suggest a high-valence configuration of the Mn4Ca complex with Mn(III)3Mn(IV) in S0, Mn(III)2Mn(IV)2 in S1, Mn(III)Mn(IV)3 in S2, and Mn(IV)4 in S3 and, thus, manganese oxidation in each of the three accessible oxidizing transitions of the water-oxidizing complex. There are no indications of formation of a ligand radical, thus rendering partial water oxidation before reaching the S4 state unlikely. The difference spectra of both manganese Kβ1,3 emission and K-edge X-ray absorption display different shapes for Mn(III) oxidation in the S2 → S3 transition when compared to Mn(III) oxidation in the S1 → S2 transition. Comparison to spectra of manganese compounds with known structures and oxidation states and varying metal coordination environments suggests a change in the manganese ligand environment in the S2 → S3 transition, which could be oxidation of five-coordinated Mn(III) to six-coordinated Mn(IV). Conceivable options for the rearrangement of (substrate) water species and metal-ligand bonding patterns at the Mn4Ca complex in the S2 → S3 transition are discussed.

Cobaloximes are popular H 2 evolution molecular catalysts, but have so far mainly been studied in non-aqueous conditions. We show here that they are also valuable for the design of artificial hydrogenases for application in neutral aqueous solutions and report on the preparation of two well-defined biohybrid species via the binding of two cobaloxime moieties {Co(dmgH) 2 } and {Co(dmgBF 2 ) 2 }(dmgH 2 = dimethylglyoxime) to apo Sperm-whale myoglobin (SwMb). All spectroscopic data confirm that the cobaloxime moieties are inserted within the binding pocket of the SwMb protein and are coordinated to a histidine residue in axial position of the cobalt complex, resulting in thermodynamically stable complexes. QC/MM docking calculations indicated coordination preference for His93 over the other histidine residue (His64) present in the vicinity. Interestingly, the redox activity of the cobalt centers is retained in both biohybrids which provides them with catalytic activity for H 2 evolution in near neutral aqueous conditions.

Ribonucleotide reduction is the only pathway for de novo synthesis of deoxyribonucleotides in extant organisms. This chemically demanding reaction, which proceeds via a carbon-centered free radical, is catalyzed by ribonucleotide reductase (RNR). The mechanism has been deemed unlikely to be catalyzed by a ribozyme, creating an enigma regarding how the building blocks for DNA were synthesized at the transition from RNA- to DNA-encoded genomes. While it is entirely possible that a different pathway was later replaced with the modern mechanism, here we explore the evolutionary and biochemical limits for an origin of the mechanism in the RNA + protein world and suggest a model for a prototypical ribonucleotide reductase (protoRNR). From the protoRNR evolved the ancestor to modern RNRs, the urRNR, which diversified into the modern three classes. Since the initial radical generation differs between the three modern classes, it is difficult to establish how it was generated in the urRNR. Here we suggest a model that is similar to the B12-dependent mechanism in modern class II RNRs.

[FeFe]-hydrogenases are known for their high rates of hydrogen turnover, and are intensively studied in the context of biotechnological applications. Evolution has generated a plethora of different subclasses with widely...