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.

The [FeFe]-hydrogenases of bacteria and algae are the most efficient hydrogen conversion catalysts in nature. Their active-site cofactor (H-cluster) comprises a [4Fe-4S] cluster linked to a unique diiron site that binds three carbon monoxide (CO) and two cyanide (CN) ligands. Understanding microbial hydrogen conversion requires elucidation of the interplay of proton and electron transfer events at the H-cluster. We performed real-time spectroscopy on [FeFe]-hydrogenase protein films under controlled variation of atmospheric gas composition, sample pH, and reductant concentration. Attenuated total reflection Fourier-transform infrared spectroscopy was used to monitor shifts of the CO/CN vibrational bands in response to redox and protonation changes. Three different [FeFe]-hydrogenases and several protein and cofactor variants were compared, including element and isotopic exchange studies. A protonated equivalent (HoxH) of the oxidized state (Hox) was found, which preferentially accumulated at acidic pH and under reducing conditions. We show that the one-electron reduced state Hred' represents an intrinsically protonated species. Interestingly, the formation of HoxH and Hred' was independent of the established proton pathway to the diiron site. Quantum chemical calculations of the respective CO/CN infrared band patterns favored a cysteine ligand of the [4Fe-4S] cluster as the protonation site in HoxH and Hred'. We propose that proton-coupled electron transfer facilitates reduction of the [4Fe-4S] cluster and prevents premature formation of a hydride at the catalytic diiron site. Our findings imply that protonation events both at the [4Fe-4S] cluster and at the diiron site of the H-cluster are important in the hydrogen conversion reaction of [FeFe]-hydrogenases.

Green algae such as Chlamydomonas reinhardtii synthesize an [FeFe] hydrogenase that is highly active in hydrogen evolution. However, the extreme sensitivity of [FeFe] hydrogenases to oxygen presents a major challenge for exploiting these organisms to achieve sustainable photosynthetic hydrogen production. In this study, the mechanism of oxygen inactivation of the [FeFe] hydrogenase CrHydA1 from C. reinhardtii has been investigated. X-ray absorption spectroscopy shows that reaction with oxygen results in destruction of the [4Fe-4S] domain of the active site H-cluster while leaving the di-iron domain (2FeH) essentially intact. By protein film electrochemistry we were able to determine the order of events leading up to this destruction. Carbon monoxide, a competitive inhibitor of CrHydA1 which binds to an Fe atom of the 2FeH domain and is otherwise not known to attack FeS clusters in proteins, reacts nearly two orders of magnitude faster than oxygen and protects the enzyme against oxygen damage. These results therefore show that destruction of the [4Fe-4S] cluster is initiated by binding and reduction of oxygen at the di-iron domain-a key step that is blocked by carbon monoxide. The relatively slow attack by oxygen compared to carbon monoxide suggests that a very high level of discrimination can be achieved by subtle factors such as electronic effects (specific orbital overlap requirements) and steric constraints at the active site.EXAFS ͉ H-cluster ͉ protein film electrochemistry ͉ biological hydrogen production ͉ green algae

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