FMN cofactor dissociation from the soluble hydrogenase of Alcaligenes eutrophus H16

The specific activity of purified soluble hydrogenase of Alcaligenes eutrophus H16 was found to vary with enzyme concentration. Specific activity as a function of concentration of purified enzyme could be fit to an equation describing the dissociation of a compound into two components. An association constant, kappa(a), was determined in this way to be 39.4 +/- 8.7 micrograms protein/ml. The concentration of the enzyme affected its kinetic parameters: a tenfold decrease in enzyme concentration caused by a reduction of the V(max) and Kappa(m) (NAD) values to 45% and 58%, respectively, of the values for undiluted (0.64 mg/ml) enzyme. Diaphorase (NAD-dependent reduction of benzyl viologen) specific activity of the hydrogenase was unaffected by dilution. The extent of dilution-induced activity loss was dependent on pH, with greater activity loss observed at higher pH values. The substrate NAD prevented loss of specific activity due to dilution, while the product NADH did not. Specific activity loss due to dilution as reversed with the addition of the cofactor FMN. Dilution of the hydrogenase caused an increase in the enzyme's specific flavin fluorescence. These results suggest that dilution of the soluble hydrogenase of Alcaligenes eutrophus causes dissociation of the cofactor FMN, and this activity loss should be taken into account as an important factor governing hydrogenase activity and kinetic properties.