Isolation and first EPR characterization of the [FeFe]-hydrogenases from green algae.
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 micromol 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.
Kamp
Christina
C
Ruhr-University Bochum, Department of Biochemistry of Plants, 44780 Bochum, Germany.
Silakov
Alexey
A
Winkler
Martin
M
Reijerse
Edward J
EJ
Lubitz
Wolfgang
W
Happe
Thomas
T
D020418:Algal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002696:Chlamydomonas / Q000201:enzymology / Q000235:genetics; D000460:Chlorophyta / Q000201:enzymology* / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006864:Hydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid
D020418:Algal Proteins; D006864:Hydrogenase