PARENT SESSION
Posters P3C C3 and Rubisco. Abstracts (617-630)

Real time assay of O₂-dependent, H₂O₂ production by spinach Rubisco. Kangmin Kim¹, Archie Portis^{*,1, 2}, ¹ Photosynthesis Research Unit, Urbana, IL, USA² Department of Crop Sciences, Urbana, IL, USA

ABSTRACT- In addition to carboxylation and oxygenation, Rubisco can also catalyze other reactions proceeding from various enzyme-bound intermediates in these two reactions. In several reports in the literature, some of the products of these reactions have been implicated in the slow decline in Rubisco activity during in vitro assay. One compound, D-glycero-2,3-pentodiulose 1,5-bisphosphate, has been shown to be a major product produced during the oxygenation of D-ribulose 1,5-bisphosphate (RuBP) by the Rhodospirillum rubrum E48Q mutant (Chen, Y.-R. and Hartman, F.C. (1995) J. Biol. Chem. 270, 11741-11744). This compound is produced by elimination of H₂O₂ from the C-2 peroxyketone derivative of RuBP, specific to the oxygenation pathway. The role of this pentodiulose bisphosphate in the decline of Rubisco activity is unclear because it is not known if there is significant formation of pentodiulose bisphosphate and H₂O₂ during catalysis by any other Rubisco. Moreover pentodiulose bisphosphate can be formed by non-enzymatic oxidation of RuBP during storage (Kane et al. (1998) Plant Physiol. 117, 1059-1069) and perhaps during long-term Rubisco assays. Using a sensitive assay, we have observed the formation of H₂O₂ in real time with spinach Rubisco. Using parallel assays, the rate of H₂O₂ formation was found to be proportional to oxygen consumption under varying CO₂ and O₂ concentrations at a constant ratio of 1:270. High temperature (38°C) increased both oxygen consumption and H₂O₂ production, resulting in a small increase in the ratio to 1:220. The observed rates of H₂O₂ production, and by inference pentodiulose bisphosphate formation, suggest that the oxygenase activity of Rubisco is a significant contributor to the observed declines in Rubisco activity with time. Our results also have implications for fully understanding the temperature responses of photosynthesis in planta.

KEY WORDS: ribulose 1,5-bisphosphate, oxidation, temperature