During SH purification, FMN-a is easily lost and can be reconstituted by adding FMN in stoichiometric amounts to the activity assay resulting in significant higher NAD + reducing activity. Experiments with separately produced hydrogenase and diaphorase modules showed that the FMN-a of hydrogenase module can be reconstituted under reducing conditions and the FMN-b of the diaphorase module is released under reducing conditions. Purified SH shows a lag phase during activity, which is explained that most of enzyme population is inactive and required for reactivation reverse electron transfer from NADH generated from an active enzyme population. In contrast to most other hydrogenases, the SH is O2 tolerant; i.e. its catalytic activity is not impaired by ambient O2. SH has been employed for the recycling of NAD(P)H in various enzymatic reactions in vitro and vivo. Compared with common NAD(P)H-regeneration systems such as formate dehydrogenase (generating CO2 as stoichiometric by-product) and glucose-6-phosphate dehydrogenase (yielding the corresponding gluconolactone), H2-driven cofactor recycling has the advantage of being 100% atom efficient, clean by-product-free and cheap since it only relies on H2 as reductant. Besides NAD + , SH is also known to reduce other electron acceptors such as ferricyanide, methylene blue and methyl viologen. This catalytic promiscuity in terms of electron acceptor may open up new avenues of H2-driven biocatalysis while avoiding costly nicotinamide cofactors. We therefore evaluated SH-driven reduction of flavins to promote a range of biocatalytic redox reactions of synthetic interest. First, we investigated the kinetic parameters for the SH-catalyzed reduction of FMN and FAD driven by H2 oxidation. The specific activity of SH for reducing FMN was 5.8 U mg -1 (20.3 s -1 ), with a Km, FMN of 680 µM (Fig. ). Adding catalytic amounts of NADH increased the FMN specific activity by 100% and led to the disappear of the lag phase. The disappearance of the lag phase is explained by faster reactivation under reducing conditions and reverse electron transfer, see above. Surprisingly, SH exhibited a significantly lower activity on FAD (0.14 U mg -1 , 0.49 s -1 ) which prohibited a precise determination of its Km value. The higher activity towards the SHrelated FMN cofactor as well as the increased FMN activity in the presence of NADH support the reaction model, that the SH is reconstituted under reducing conditions by free oxidized flavin. After reduction of bound flavin, it is released again (Fig. ). The FMN-b in the Rossmann fold is deeper submerged than the binding site of NAD + as seen in the related SH from Hydrogenophilus thermoluteolus , explaining the 17 times lower specific activities in comparison to NAD + as the substrate. The Fe-S clusters of the SH are burrowed in the protein Tab. 1 H2-driven asymmetric reduction of alkenes Encouraged by this result, we explored the H2-driven regeneration of reduced flavins to drive biocatalytic redox reactions (Fig. ). As a first model reaction, we chose the stereoselective reduction of activated C=C-double bonds catalyzed by the ene reductase Old Yellow Enzyme from Thermus scotoductus (TsOYE). In accordance with the substrate spectrum of TsOYE, a range of cyclic enones was reduced (Tab. 1). Control reactions in the absence of either SH or H2 under otherwise identical conditions gave no detectable conversion. In particular, ketoisophorone was fully converted (99% conversion, Tab. 1 entry 1), albeit yielding product with a significantly reduced optical purity (37%). Similar observations had been made with this product and are generally associated with racemization under the reaction conditions (Tab. S3). Interestingly, the control reaction without FMN gave a low but detectable conversion, which may be indicative of a slow, direct electron transfer from SH to the flavin prosthetic group in TsOYE. Pleasingly, already under non-optimized conditions, significant catalytic performance for both SH and FMN of up to 8400 and 105, respectively, were observed (Tab.