Isolation and first EPR characterization of the [FeFe]-hydrogenases from green algae

Christina Kamp, Alexey Silakov, Martin Winkler, Edward J. Reijerse, Wolfgang Lubitz, Thomas Happe

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102 Scopus citations


Hydrogenase expression in Chlamydomonas reinhardtii can be artificially induced by anaerobic adaptation or is naturally established under sulphur deprivation. In comparison to anaerobic adaptation, sulphur-deprived algal cultures show considerably higher expression rates of the [FeFe]-hydrogenase (HydA1) and develop a 25-fold higher in vitro hydrogenase activity. Based on this efficient induction principle we have established a novel purification protocol for the isolation of HydA1 that can also be used for other green algae. From an eight liter C. reinhardtii culture 0.52 mg HydA1 with a specific activity of 741 μmol H2 min- 1 mg- 1 was isolated. Similar amounts were also purified from Chlorococcum submarinum and Chlamydomonas moewusii. The extraordinarily large yields of protein allowed a spectroscopic characterization of the active site of these smallest [FeFe]-hydrogenases for the first time. An initial analysis by EPR spectroscopy shows characteristic axial EPR signals of the CO inhibited forms that are typical for the Hox-CO state of the active site from [FeFe]-hydrogenases. However, deviations in the g-tensor components have been observed that indicate distinct differences in the electronic structure between the various hydrogenases. At cryogenic temperatures, light-induced changes in the EPR spectra were observed and are interpreted as a photodissociation of the inhibiting CO ligand.

Original languageEnglish (US)
Pages (from-to)410-416
Number of pages7
JournalBiochimica et Biophysica Acta - Bioenergetics
Issue number5
StatePublished - May 2008

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Cell Biology


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