The source and characteristics of chemiluminescence associated with the oxygenase reaction catalyzed by Mn(2+)-ribulosebisphosphate carboxylase.
We confirm the observation of Mogel and McFadden (Mogel, S.N., and McFadden, B. A. (1990) Biochemistry 29, 8333-8337) that ribulosebisphosphate carboxylase/oxygenase (rubisco) exhibits chemiluminescence while catalyzing its oxygenase reaction in the presence of Mn2+. However, our results with the spinach and Rhodospirillum rubrum enzymes differ markedly in the following respects. 1) Chemiluminescence intensity was directly proportional to enzyme concentration and behaved as if representing the rate of oxygenase catalysis. 2) The wavelength spectrum peaked at about 770 nm and extended beyond 810 nm. This seems inconsistent with chemiluminescence generated by simultaneous decay of pairs of singlet O2 molecules. It is consistent with manganese(II) luminescence and we discuss its possible sources. The time course of chemiluminescence (resolution, 0.25 s) was distinctively different for spinach and R. rubrum enzymes during the initial 5 s of catalysis, with the bacterial enzyme exhibiting a pronounced initial "burst." Chemiluminescence by the spinach enzyme responded to substrate concentrations in a manner consistent with known oxygenase properties, exhibiting Michaelis-Menten kinetics with ribulose-1,5-bisphosphate (Km 400 nM). Chemiluminescence required carbamylated enzyme with Mn2+ bound at the active site (activation energy, -57.1 KJ.mol-1). As an indicator of oxygenase activity, chemiluminescence represents an improvement over oxygen electrode measurements in response time and sensitivity by factors of at least 100.
Lilley
R M
RM
Department of Biological Sciences, University of Wollongong, New South Wales, Australia.
Riesen
H
H
Andrews
T J
TJ
D007700:Kinetics; D008163:Luminescent Measurements; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000378:metabolism* / Q000494:pharmacology; D010100:Oxygen / Q000494:pharmacology; D010105:Oxygenases / Q000378:metabolism*; D010944:Plants / Q000201:enzymology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012273:Ribulose-Bisphosphate Carboxylase / Q000302:isolation & purification / Q000378:metabolism*; D013053:Spectrophotometry; D013816:Thermodynamics
D011994:Recombinant Proteins; D008345:Manganese; D010105:Oxygenases; D012273:Ribulose-Bisphosphate Carboxylase; D008274:Magnesium; D010100:Oxygen