Fig. 4.

Purified synthetic [FeFe]-hydrogenase displays hydrogenase activity in vitro. E. coli strain PK4854 was co-transformed with pUNI-Tte-Hydhisc and one of three accessory plasmids: pSU-Sh-EFG; pSU23-Sh-GX-EF; or pSUtat-Sh-GX-EF, as indicated. Harvested cells were lysed either by sonication or using a chemical cocktail (BPER, Thermo Scientific) as indicated and enzyme isolated by IMAC. ‘Proton reduction’ activity involved methyl viologen-dependent H₂ production measured in a modified Clark-type electrode. The reaction was initiated by the addition of 10 μg of purified enzyme. ‘H₂ oxidation’ assays involved H₂-dependent benzyl viologen reduction monitored at 578 nm in a UV-vis spectrometer. The reaction was started by the addition of 20 μg of purified enzyme and recorded at 37 °C. Error bars represent the standard error of three independent experiments.