Influence of the [4Fe-4S] cluster coordinating cysteines on active site maturation and catalytic properties of C. reinhardtii [FeFe]-hydrogenase.

[FeFe]-Hydrogenases catalyze the evolution and oxidation of hydrogen using a characteristic cofactor, termed the H-cluster. This comprises an all cysteine coordinated [4Fe-4S] cluster and a unique [2Fe] moiety, coupled together via a single cysteine. The coordination of the [4Fe-4S] cluster in HydA1 from Chlamydomonas reinhardtii was altered by single exchange of each cysteine (C115, C170, C362, and C366) with alanine, aspartate, or serine using site-directed mutagenesis. In contrast to cysteine 115, the other three cysteines were found to be dispensable for stable [4Fe-4S] cluster incorporation based on iron determination, UV/vis spectroscopy and electron paramagnetic resonance. However, the presence of a preformed [4Fe-4S] cluster alone does not guarantee stable incorporation of the [2Fe] cluster. Only variants C170D, C170S, C362D, and C362S showed characteristic signals for an inserted [2Fe] cluster in Fourier-transform infrared spectroscopy. Hydrogen evolution and oxidation were observed for these variants in solution based assays and protein-film electrochemistry. Catalytic activity was lowered for all variants and the ability to operate in either direction was also influenced.