Chemiluminescence of Mn2+-activated Rubisco: temperature and pH responses differ between L-2 and L8S8 forms, and inhibitors provide no evidence for involvement of active oxygen species

Chemiluminescence associated with the oxygenase activity of Mn2+-activated D-ribulose-1,5- bisphosphate carboxylase/oxygenase (Rubisco) was investigat ed using the L8S8 enzymes from spinach and Synechococcus PCC6301, and the L -2 enzyme from Rhodospirillum rubrum. Chemiluminescence was measured with a luminometer and oxygenase activity with an oxygen electrode. The relative luminescence yield (light emitted per oxygenase turnover) in the steady-sta te at pH 8.1 was highest with spinach Rubisco; the enzymes from Synechococc us and R. rubrum exhibited approximately one half and one fifth, respective ly, of the spinach value. The relative luminescence yield from the L8S8 enz ymes was unaffected by temperature (20-40 degrees C) and pH (7.6-9.0). In c ontrast, the luminescence yield of R. rubrum Rubisco varied with temperatur e and pH, and its O-2-consuming activity exhibited a lower activation energ y than that of the L8S8 enzymes. The singlet O-2-reactive compounds diazabi cyclo[ 2.2.2] octane and 10,10-dimethyl-9,9-biacridinium dinitrate (lucigen in) had no effect on chemiluminescence. Other compounds tested inhibited ch emiluminescence but also inhibited O-2 consumption. The inhibition of chemi luminescence of spinach Rubisco required several seconds to exert its maxim al effect, implying that the inhibitors had access to the active site only at a particular stage of the catalytic cycle. The data are consistent with the Mn2+ ion at the active site being the source of the luminescence.