Spectroelectrochemical characterization of the active site of the [FeFe] hydrogenase HydA1 from Chlamydomonas reinhardtii

Alexey Silakov, Christina Kamp, Eduard Reijerse, Thomas Happe, Wolfgang Lubitz

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Abstract

Hydrogenases catalyze the reversible oxidation of molecular hydrogen. The active site of the [FeFe] hydrogenases (H-cluster) contains a catalytically active binuclear subcluster ([2Fe]H) connected to a "cubane" [4Fe4S]H subcluster. Here we present an IR spectroelectrochemical study of the [FeFe] hydrogenase HydA1 isolated from the green alga Chlamydomonas reinhardtii. The enzyme shows IR bands similar to those observed for bacterial [FeFe] hydrogenases. They are assigned to the stretching vibrations of theCN- andCO ligands on both irons of the [2Fe] H subcluster. By following changes in frequencies of the IR bands during electrochemical titrations, two one-electron redox processes of the active enzyme could be distinguished. The reduction of the oxidized state (Hox) occurred at a midpoint potential of -400 mV vs NHE (H ox/Hred transition) and relates to a change of the formal oxidation state of the binuclear subcluster. A subsequent reduction (H red/Hsred transition) was determined to have a midpoint potential of -460 mV vs NHE. On the basis of the IR spectra, it is suggested that the oxidation state of the binuclear subcluster does not change in this transition. Tentatively, a reduction of the [4Fe4S]H cluster has been proposed. In contrast to the bacterial [FeFe] hydrogenases, where the bridgingCOligand becomes terminal when going from Hox to H red, in HydA1 the bridging CO is present in both the Hox and Hred state. The removal of the bridging CO moiety has been observed in the Hred to Hsred transition. The significance of this result for the hydrogen conversion mechanism of this class of enzymes is discussed.

Original languageEnglish (US)
Pages (from-to)7780-7786
Number of pages7
JournalBiochemistry
Volume48
Issue number33
DOIs
StatePublished - Aug 25 2009

All Science Journal Classification (ASJC) codes

  • Biochemistry

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