Chemiluminescence of Mn 2+-activated Rubisco: temperature and pH responses differ between L2 and L8S8 forms, and inhibitors provide no evidence for involvement of active oxygen species

Abstract

Chemiluminescence associated with the oxygenase activity of Mn ²⁺ -activated D-ribulose-1,5- bisphosphate carboxylase/oxygenase (Rubisco) was investigated using the L ₈ S ₈ enzymes from spinach and Synechococcus PCC6301, and the L ₂ enzyme from Rhodospirillum rubrum. Chemi-luminescence 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 L ₈ S ₈ 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 O ₂ -consuming activity exhibited a lower activation energy than that of the L ₈ S ₈ enzymes. The singlet O ₂ -reactive compounds diazabi-cyclo[ 2.2.2]octane and 10,10′-dimethyl-9,9′-biacridinium dinitrate (lucigenin) had no effect on chemi-luminescence. Other compounds tested inhibited chemiluminescence but also inhibited O ₂ 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 Mn ²⁺ ion at the active site being the source of the luminescence.