In vitro Maturation of Hydrogenases

In vitro maturation of hydrogenases was achieved by incubating 800 ng apo-protein (0.04 μM HYDA1) under strictly anaerobic conditions in 400 μl of 0.1 M potassium phosphate buffer, pH 6.8, with 2 mM sodium dithionite (NaDT) at 25°C for 30 min with a 10-fold molar excess of [2Fe]^MIM if not stated otherwise. Subsequent in vitro activity measurements using NaDT-reduced methyl viologen as artificial electron donor were done as previously described³³ . Maturation of HYDA1^MIM for EPR and FTIR measurements was carried out with 150 μM apoHYDA1. The protein was subsequently purified and re-buffered to 0.01 M Tris-HCl, pH 8.0, 2 mM NaDT, by size exclusion chromatography using a NAP™ 5 column (GE Healthcare) and concentrated to 500 μM for EPR and FTIR measurements using Amicon Ultra centrifugal filters 10K (Millipore). Spectra of 12 mM [2Fe]^MIM were recorded in 20 mM HEPES buffer pH 7.5, 100 mM KCl.