Molecular and Biochemical Basis for Stable Carbon Isotope Ratios in Marine Autotrophs using Form IA RubisCO

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Stable carbon isotope data are used widely in ecology and earth sciences. These data on isotope fractionations influence our understandings of processes occurring on molecular to global scales. Hindcasts of past climates, models constraining the fates of present day greenhouse gas emissions, inferences of trophic transfers within complex ecosystems, and elucidation of enzyme and cellular controls on primary productivity have all used stable carbon isotope fractionation data (delta 13C values). Despite the wide applicability of such fractionation information, such data are nearly always interpreted assuming carbon is fixed and its isotopes fractionated as if the ribulose?1,5?bisphosphate?carboxylase/oxygenase (RubisCO) of spinach was responsible! This is probably a huge mistake and may substantially invalidate all of the subsequent data interpretations for problems such as those mentioned above.In the few cases that have been studied, the kinetic isotope effects (KIEs) forRubisCOs, the degree to which the delta 13C of the carbon fixed differs from the C02 substrate used, have been demonstrated to vary broadly. Thus, determining the KIEs of different RubisCOs is critical to understanding, delta 13C interpretation, and modeling of delta 13C values in oceanography. As a result, it is a long?term research goal to determine KIEs from all forms of RubisCO in autotrophs. In order to determine the KIEs of Form IA RubisCOs, these carbon-fixing enzymes will be isolated and purified from representative marine picophytoplankton and chemoautotrophic bacteria. Model species Synechococcus WH7803, Prochlorococcus MED 4, and two sulfur?oxidizing chemoautotrophs, symbionts of the coastal and vent bivalves, Solemya velum and Bathymodiolus thermophilus will be used. The isolated proteins will be characterized with respect to amino acid sequence, composition, Vmax, and Km (CO2). Finally, the kinetic isotope effects will be determined by high precision methods. Determining the KIEs for Form IA RubisCOs will allow us to evaluate a fundamental assumption of delta 13C analyses, and to infer the factors affecting oceanic primary productivity with a substantial improvement in accuracy.