Alexey Silakov

A Model of the [FeFe] Hydrogenase Active Site with a Biologically Relevant Azadithiolate Bridge: A Spectroscopic and Theoretical Investigation

Convincing evidence for the presence of a nitrogen atom in the dithiolate bridge of the active site of native [FeFe] hydrogenases (B) is provided by a spectroscopic, electrochemical, and theoretica ...

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.

The [FeFe]‐hydrogenase maturase HydF from Clostridium acetobutylicum contains a CO and CN− ligated iron cofactor

Biosynthesis of the [FeFe] hydrogenases active site (H‐cluster) requires three maturation factors whose respective roles are not understood yet. The clostridial maturation enzymes (CaHydE, CaHydF and CaHydG) were homologously overexpressed in their native host Clostridium acetobutylicum. CaHydF was able to activate Chlamydomonas reinhardtii [FeFe] hydrogenase apoprotein (CrHydA1apo) to almost 100% compared to the native specific hydrogen evolution activity. Based on electron paramagnetic resonance spectroscopy and Fourier‐transform infrared spectroscopy data the existence of a [4Fe4S] cluster and a CO and CN− ligand coordinated di‐iron cluster is suggested. This study contains the first experimental evidence that the bi‐nuclear part of the H‐cluster is assembled in HydF.