Investigating [NiFe]-hydrogenases in γ-Proteobacteria

  • Alexander Finney

Student thesis: Doctoral ThesisDoctor of Philosophy


A multitude of microorganisms possess the ability to metabolise molecular hydrogen (H2). The major enzyme family involved in hydrogen metabolism are Hydrogenases. These enzymes catalyse the reversible conversion of molecular hydrogen to protons and electrons (H2 ↔ 2H+ + 2e-). These enzymes have the potential to be utilised for biotechnological applications such as hydrogen fuel cells, but they also represent promising drug targets for inhibition of bacterial energy metabolism both within the gastrointestinal tract and after infection. Therefore, further understanding and discoveries made in the hydrogenase field warrants progression into applied medical and biotechnological research areas. Hydrogenases are also interesting due to their phylogeny and physiology in a large number of microbial species. These enzymes are categorised by their active site architecture. One well studied, ancient group is termed the [NiFe]-hydrogenases, which all harbour a complex NiFe(CN-)2CO active site in the ‘large’ catalytic subunit and usually have three iron-sulfur clusters within a ‘small’ electron transferring partner subunit. [NiFe]-hydrogenases have undergone massive diversification, with four major phylogenetic subgroups arising. The major part of this Thesis concerns work on a Group 4 [NiFe]-hydrogenase that functions in partnership with a formate dehydrogenase as a formate hydrogenlyase (FHL). This FHL complex generates H2 and CO2 from the disproportionation of formate (CHOO- + H+ ↔ H2 + CO2). In this Thesis, genetic and biochemical characterisation of Pectobacterium atrosepticum SCRI1043, a potato pathogen, led to the identification of a novel FHL complex. The [NiFe]-hydrogenase in this organism is similar to that of Escherichia coli Hydrogenase-4, with an extended membrane domain similar to that of respiratory Complex I. Importantly, the P. atrosepticum formate dehydrogenase is selenium-free, while previously characterised FHL complexes have selenocysteine-containing formate dehydrogenases. Using genetic and biochemical approaches it was shown that the [NiFe]-hydrogenase and a formate dehydrogenase were vital for H2 production by P. atrosepticum. Using plant infection assays it was also shown that the gene encoding the formate dehydrogenase was important for full infective ability of P. atrosepticum in potato plants and tubers. The latter part of this Thesis focuses on developing genetic tools to study this novel FHL from P. atrosepticum as well as Hydrogenase-1 and -2 from E. coli.
Date of Award2019
Original languageEnglish
SupervisorFrank Sargent (Supervisor) & Sarah Coulthurst (Supervisor)


  • [NiFe]-hydrogenase
  • Pectobacterium
  • Formate Hydrogenlyase
  • Formate Dehydrogenase

Cite this