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

Research output: Contribution to journalArticle

85 Citations (Scopus)

Abstract

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
Volume1777
Issue number5
DOIs
StatePublished - May 1 2008

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Hydrogenase
Chlorophyta
Algae
Paramagnetic resonance
Carbon Monoxide
Chlamydomonas reinhardtii
Sulfur
Catalytic Domain
Photodissociation
Chlamydomonas
Cryogenics
Electronic structure
Purification
Tensors
Spectrum Analysis
Spectroscopy
Ligands
Light
Temperature
Proteins

All Science Journal Classification (ASJC) codes

  • Biophysics
  • Biochemistry
  • Cell Biology

Cite this

Kamp, Christina ; Silakov, Alexey ; Winkler, Martin ; Reijerse, Edward J. ; Lubitz, Wolfgang ; Happe, Thomas. / Isolation and first EPR characterization of the [FeFe]-hydrogenases from green algae. In: Biochimica et Biophysica Acta - Bioenergetics. 2008 ; Vol. 1777, No. 5. pp. 410-416.
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Isolation and first EPR characterization of the [FeFe]-hydrogenases from green algae. / Kamp, Christina; Silakov, Alexey; Winkler, Martin; Reijerse, Edward J.; Lubitz, Wolfgang; Happe, Thomas.

In: Biochimica et Biophysica Acta - Bioenergetics, Vol. 1777, No. 5, 01.05.2008, p. 410-416.

Research output: Contribution to journalArticle

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