FIGURE 4.

H₂ generation in reconstitution assays of recombinant C. reinhardtii PFR1 and [Fe-Fe]-hydrogenases. A, each reaction contained PFR1 (0.7 μm) and 0.01 μm HYDA1 of C. reinhardtii (except one reaction, which contained C. acetobutylicum ferredoxin (CAC0303) and [Fe-Fe]-hydrogenase HYDA, indicated by the label CaHYDA), 10 mm pyruvate, and 2 mm CoA in 100 mm potassium phosphate buffer, pH 6.8. Electron carriers were applied as indicated below the x axis (10 mm methyl viologen (MV), 40 μm of the Chlamydomonas ferredoxins PETF, FDX2, or FDX5, or 40 μm clostridial ferredoxin CAC0303). The reaction mixtures were incubated for 30 min at 37 °C before analyzing the amount of H₂ in the gas phase. As controls, reaction mixtures were analyzed that lacked one of the enzymes or proteins indicated by a dash. B, the dependence of PFR1-coupled H₂ generation on PETF concentration was determined using C. reinhardtii HYDA1 in reaction mixtures as described for A and the indicated concentrations of PETF. The values and standard deviations shown in all of the experiments are from three independent PFR1 preparations, and the H₂ production rate was related to mg of PFR1 enzyme. The error bars indicate the standard deviation.