Chemiluminescence of Mn²⁺-activated Rubisco: Temperature and pH responses differ between L₂ 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 investigated using the L₈S₈ enzymes from spinach and Synechococcus PCC6301, and the L₂ 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-state at pH 8.1 was highest with spinach Rubisco; the enzymes from Synechococcus and R. rubrum exhibited approximately one half and one fifth, respectively, of the spinach value. The relative luminescence yield from the L₈S₈ enzymes was unaffected by temperature (20-40°C) and pH (7.6-9.0). In contrast, the luminescence yield of R. rubrum Rubisco varied with temperature and pH, and its O₂-consuming activity exhibited a lower activation energy than that of the L₈S₈ enzymes. The singlet O₂-reactive compounds diazabicyclo[2.2.2]octane and 10,10'-dimethyl-9,9'-biacridinium dinitrate (lucigenin) had no effect on chemiluminescence. Other compounds tested inhibited chemiluminescence but also inhibited O₂ consumption. The inhibition of chemiluminescence of spinach Rubisco required several seconds to exert its maximal 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.