Spectroscopic studies of chemiluminescence by Mn++-activated rubisco: Singlet oxygen is entirely absent and the emission spectrum differs between forms of rubisco.

Abstract

The manganese ion at the active site of Mn-activated Rubisco luminesces while the enzyme is catalysing the oxygenation of ribulose bisphosphate. Since luminescence in other systems is sometimes indicative of the involvement of singlet O2, we sought to detect the infrared emission at 1268 nm characteristic of the decay of monomolecular singlet O2 to the triplet state. Total luminescence emitted during oxygenation was monitored simultaneously with a photomultiplier (sensitive to wavelengths from 300 - 800 nm) and a cooled germanium detector (1.1 to 1.7 mm) using filters to select various ranges of wavelengths. When chemiluminescence was monitored by the photomultiplier, no emission >1100 nm was seen by the Ge detector, even in 2H2O solvent, precluding the involvement of singlet O2. The calculated upper limit for any radiation with wavelengths longer than 1100 nm was less than 10-6 the intensity of radiation at shorter wavelengths. However, luminescence >1100 nm was readily detected with the lactoperoxidase system that is known to generate singlet O2. An f4 0.5m spectrograph utilising a back-thinned CCD detector and low dispersion grating blazed at 800nm was constructed. This system could accumulate an entire spectrum in a few seconds. The spectra of chemiluminescence by Mn++-activated rubisco were determined for the enzyme from a range of organisms. All exhibited smooth, broad emission (~150 nm FWHM) characteristic of Mn++-luminescence, peaking between 797 and 800 nm for L8S8 Rubiscos (tobacco and spinach) but at 760 nm for L2 (bacterial) Rubisco.