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) ...

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Super‐reduziert und super‐aktiv: Ein neuer Redoxzustand des aktiven Zentrums der [FeFe]‐Hydrogenasen von Grünalgen wurde identifiziert und mittels EPR‐ und FTIR‐Spektroskopie charakterisiert. Elektrochemische und In‐vitro‐Versuche zeigen, dass diese Form eine hohe Wasserstoffproduktionsrate aufweist, was vermuten lässt, dass es sich um einen Zwischenzustand im katalytischen Zyklus aller [FeFe]‐Hydrogenasen handelt.