International Journal of Hydrogen Energy 27 (2002) 1413–1420 www.elsevier.com/locate/ijhydene How bacteria get energy from hydrogen: a genetic analysis of periplasmic hydrogen oxidation in Escherichia coli AlexandraDubini a ,RachaelL.Pye a ,RachaelL.Jack a; b ,TracyPalmer a; b , FrankSargent a ; * a Centre for Metalloprotein Spectroscopy and Biology, School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom b Department of Molecular Microbiology, John Innes Centre, Norwich NR4 7UH, United Kingdom Abstract Dihydrogen oxidation is an important feature of bacterial energy conservation. In Escherichia coli hydrogen oxidation (‘uptake’) is catalysed by membrane-bound [NiFe] hydrogenase-1 and [NiFe] hydrogenase-2. The bulk of these uptake isoenzymes is exposed to the periplasm and biosynthesis of the proteins involves membrane transport via the twin-arginine translocation(Tat)pathway.Hydrogenase-2isencodedbythe hybOABCDEFG operonandthecoreenzymeisaheterodimer of HybO and HybC. HybO is synthesised with a twin-arginine signal peptide. HybOC is associated with two other proteins (HybA and HybB) that complete the respiratory complex. The HybOC dimer is bound to the cytoplasmic membrane and appears to be anchored via a hydrophobic transmembrane -helix located at the C-terminus of HybO. Thus, hydrogenase-2 is an example of an integral membrane protein assembled in a Tat-dependent (Sec-independent) manner. Studies of the biosynthesis, targeting, and assembly of hydrogenase-2 would set a paradigm for all respiratory complexes of this type. ? 2002 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved. Keywords: Escherichia coli; Hydrogenase; Metallenzyme biosynthesis 1. Introduction Hydrogengasisanimportantsourceofreductantutilised by a wide range of bacteria. For example, the hydroge- nase enzymes that catalyse hydrogen oxidation are present in photosynthetic bacteria (Rhodobacter), nitrogen xers (Azotobacter, Rhizobium), cyanobacteria (Synechocys- tis), strict anaerobes (Clostridia), and Salmonella and Escherichia coli species [1]. E. coli itself is not capable of autotrophic growth with carbon dioxide, neither does it photosynthesise or x molecular nitrogen. This not with- standing, however, E. coli displays an otherwise quite remarkable exibility in its ability to adapt and survive in a variety of dierent growth conditions. Depending ∗ Corresponding author. Tel.: +44-1603-592-889; fax: +44-1603-592-250. E-mail address: f.sargent@uea.ac.uk (F. Sargent). on prevailing environmental conditions oxygen, nitrate, fumarate, trimethylamine N-oxide (TMAO), dimethyl sulfoxide (DMSO), and nitrite can act as terminal electron acceptors while NADH, formate, glycerol-3-phosphate, succinate,lactate,pyruvate,andhydrogencanallbeutilised as respiratory electron donors [2]. E. coli K-12 has two modes of hydrogen metabolism— respiratory hydrogen oxidation (“uptake”) linked to quinone reduction, and non-energy conserving hydro- gen evolution during fermentative growth [3]. Of the four hydrogenase isoenzymes expressed in E. coli, two (Hydrogenase-1 and -2) have been shown to be involved in periplasmic hydrogen uptake, while the re- maining enzymes (hydrogenase-3 and -4) are part of cy- toplasmically oriented formate hydrogenlyase complexes [1,3]. The respiratory uptake isoenzymes hydrogenase-1 and hydrogenase-2 are multi-subunit, membrane-bound, nickel-containing Fe–S proteins [1]. The bulk of these up- take enzymes, including the subunits binding the special 0360-3199/02/$22.00 ? 2002 International Association for Hydrogen Energy. Published by Elsevier Science Ltd. All rights reserved. PII:S0360-3199(02)00112-X