Purification and characterization of [Fe]-hydrogenase from high yielding hydrogen-producing strain, Enterobacter cloacae IIT-BT08 (MTCC 5373) Tumpa Dutta*, Amit Kumar Das, Debabrata Das Department of Biotechnology, Indian Institute of Technology, Kharagpur, WB 721302, India article info Article history: Received 30 July 2008 Received in revised form 14 May 2009 Accepted 15 May 2009 Available online 17 June 2009 Keywords: Fe-hydrogenase Hydrogen evolution Fe–S protein Ferredoxin abstract Fe-hydrogenase from Enterobacter cloacae IIT-BT08 was purified 1284 fold with specific activity of 335 mmol H 2 /min/mg protein for hydrogen evolution using reduced methyl viologen as an electron-donor at 25  C. The molecular weight of the monomeric enzyme was determined to be 51 kDa by MALDI-ToF mass spectrometry. The PI of the enzyme was w5.6 displaying its acidic nature. The optimal temperature and pH for hydrogen evolution was 37  C and 7–7.2 respectively. The affinity constant, K m for reduced methyl viologen was 0.57  0.03 mM and that of reduced ferredoxin was 0.72  0.04 mM. The enzyme contained w11.47 gm-atom Fe/mol of Fe-hydrogenase. Electron paramagnetic resonance analysis ascertained the existence of iron molecules as [4Fe–4S] clusters. The internal amino acid sequences of trypsin digested peptides of hydrogenase as determined by ESI MS/MS Q-ToF showed 80-87% identities with the respective sequences of Clostridium sp. and Trichomonas sp. hydrogenase. ª 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. 1. Introduction Hydrogenases (H 2 ase) are metallo-redox enzymes catalyzing the reversible conversion of hydrogen into its constituents, two protons and two electrons. 2H þ þ 2e  H 2 ase 4 H 2 The enzyme was first discovered by Stephenson and Stick- land in prokaryotes [1].H 2 ases are heterogeneous group of enzymes with different sizes, subunit compositions and cellular localizations. On the basis of metal content of the catalytic subunit, H 2 ase can be classified into three categories – those containing only Fe at the active site, called Fe-H 2 ase (H 2 evolution) and those with Ni, Fe and sometimes Se, [Ni–Fe]/[Ni–Fe–Se] H 2 ase (H 2 uptake) and metal free H 2 ase [2]. The presence of the metal ion varies with respect to the cellular localization, metabolic function and specificity of the redox partner(s) [3,4]. The metal ions stabilize the enzyme at low oxidation state and thus help in the transfer of electrons to its respective redox partner. The oxidation rate decreases in the series Fe > Ni with respect to pH [2,4].H 2 ases are involved directly or indirectly in energy metabolism. The presence of Fe-H 2 ase has been identified both in aerobic and anaerobic prokaryotes and eukaryotes [3]. The function of the cyto- plasmic enzyme is to remove the excess reducing equivalents of the cells during microbial fermentations and that of the periplasmic enzymes in hydrogen oxidation [4,5]. Compelling evidences from x-ray crystallography and spectroscopic studies indicate that the C-terminal region of Fe-H 2 ase * Corresponding author. Tel.: þ91 9433234985. E-mail address: tumpa.dutta@gmail.com (T. Dutta). Available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/he 0360-3199/$ – see front matter ª 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijhydene.2009.05.076 international journal of hydrogen energy 34 (2009) 7530–7537