[FeFe] hydrogenases catalyze the reversible reduction of protons to molecular hydrogen (Adams (1990); Biochim Biophys Acta 1020(2): 115-145) and are of significant interest for the biological production of hydrogen fuel. They are complex proteins with active sites containing iron, sulfur, and carbon monoxide and cyanide ligands (Peters et al. (1998); Science 282(5395): 1853-1858). Maturation enzymes for [FeFe] hydrogenases have been identified (Posewitz et al. (2004); J Biol Chem 279(24): 25711-25720), but complete mechanisms have not yet been elucidated. The study of [FeFe] hydrogenases has been impeded by the lack of an easily manipulated expression/activation system capable of producing these complex and extremely oxygen-sensitive enzymes. Here we show the first expression of functional [FeFe] hydrogenases in an Escherichia coli-based cell-free transcription/translation system. We have produced and matured both algal and bacterial hydrogenases using E. coli cell extracts containing the HydG, HydE, and HydF proteins from Shewanella oneidensis. The current system produces approximately 22 microg/mL of active protein, constituting approximately 44% of the total protein produced. Active protein yield is greatly enhanced by pre-incubation of the maturation enzyme-containing extract with inorganic iron and sulfur for reconstitution of the [Fe-S] clusters in HydG, HydE, and HydF. The absence of cell walls permits direct addition of cofactors and substrates, enabling rapid production of active protein and providing control over the maturation conditions. These new capabilities will enhance the investigation of complex proteins requiring helper proteins for maturation and move us closer to the development of improved hydrogenases for biological production of hydrogen as a clean, renewable alternative fuel.
The model iron-sulfur (Fe-S) protein ferredoxin (Fd) from Synechocystis sp. PCC 6803 has been simultaneously produced and matured in a cell-free production system. After 6 h of incubation at 37 degrees C, Fd accumulated to >450 microg/mL. Essentially all was soluble, and 85% was active. Production and maturation of the protein in the cell-free system were found to be dependent in a coupled manner on the concentration of the supplemented iron and sulfur sources, ferrous ammonium sulfate and cysteine, respectively. The recombinant expression of ISC helper proteins during cell extract preparation did not increase cell-free Fd accumulation or activity, although the efficiency of iron and cysteine utilization increased. Fd maturation was independent of protein production rate, and proceeded at a constant rate throughout the period of active translation. In addition, incubation of denatured apo Fd with cell-free reaction components resulted in recovery of Fd activity, supporting the interpretation that maturation mechanisms did not act co-translationally. Incubation at 28 degrees C increased total and active protein accumulation, but decreased the ratio of active to total Fd produced. In summary, the high product yields and folding efficiency make the cell-free system described here an attractive platform for the study of Fe-S protein production and maturation. The system enables both small-volume, high throughput investigations as well as larger scale production. To our knowledge, this is the first demonstration of directed, high-yield production and maturation of an Fe-S protein in a cell-free system.
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