5 M click here ammonium sulfate and loaded onto a hydrophobic interaction chromatography column (Phenyl-Sepharose HiLoad; 2.6 × 10 cm) equilibrated with 0.5 M ammonium sulfate in buffer A. Protein was eluted using a stepped ammonium sulfate gradient (60 ml each of 0.4 M, 0.3 M, 0.2 M, 0.1 M and without

ammonium sulfate) in buffer A and at a flow rate of 5 ml min-1. The hydrogen-oxidizing activity was recovered in the fractions eluting with only buffer A. Fractions containing enzyme activity were concentrated by centrifugation at 7,500 × g in centrifugal filters (Amicon Ultra, 50 K, Millipore, Eschborn, Germany) and applied to a Hi-Load Superdex-200 gel filtration column (2.6 × 60 cm) equilibrated with buffer A containing 0.1 M NaCl. Fractions containing the hydrogen-oxidizing activity eluted after 47 ml (peak maximum); the void volume Vo of the column was 45 ml and the separation range was from 60-600 kDa. Protein was stored in buffer A containing 0.1 M NaCl at a concentration Akt inhibitor of 3 mg protein ml-1. The activity

was stable for several months when stored at -80°C. Mass spectrometric identification of proteins For mass spectrometric analysis the gel band showing H2: BV oxidoreductase activity after hydrophobic interaction chromatography was excised and the proteins within the band were in-gel digested following standard protocols [37]. Briefly, protein disulfides were reduced with DTT and cysteines those were alkylated with iodoacetamide. Digestion was performed at 37°C for two hours using trypsin as protease. ProteaseMax® surfactant was used in the digestion and extraction solutions to improve the recovery of hydrophobic peptides. The peptide extracts were analyzed by LC/MS on an UltiMate Nano-HPLC system (LC Packings/Dionex) coupled to an LTQ-Orbitrap XL mass spectrometer (ThermoFisher Scientific) equipped with a Salubrinal cell line nanoelectrospray ionization source (Proxeon). The samples were loaded onto a trapping column (Acclaim PepMap C18, 300 μm × 5 mm, 5 μm, 100Å, LC Packings) and washed for 15 min with 0.1% trifluoroacetic acid at a flow rate of 30 μl/min. Trapped peptides were eluted using a separation column (Acclaim

PepMap C18, 75 μm × 150 mm, 3 μm, 100Å, LC Packings) that had been equilibrated with 100% A (5% acetonitrile, 0.1% formic acid). Peptides were separated with a linear gradient: 0-50% B (80% acetonitrile, 0.1% formic acid) in 90 min, 50-100% B in 1 min, remain at 100% B for 5 min. The column was kept at 30°C and the flow-rate was 300 nl/min. During the duration of the gradient, online MS data were acquired in data-dependent MS/MS mode: Each high-resolution full scan (m/z 300 to 2000, resolution 60,000) in the orbitrap analyzer was followed by five product ion scans (collision-induced dissociation (CID)-MS/MS) in the linear ion trap for the five most intense signals of the full scan mass spectrum (isolation window 2 Th). Both precursor and fragment ions were analyzed in the orbitrap analyzer.