Molecular and Biochemical Basis for Stable Carbon Isotope Ratios in Marine Autotrophs Using Form IC and ID 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 (d13C values). RubisCO, the CO2-fixing enzyme for the Calvin Cycle, is by far the most important enzyme catalyzing carbon-fixation on earth, used widely by photoautotrophs and chemoautotrophs. Presently, five forms of RubisCO have been described in autotrophs: Forms IA-D, and Form II, which differ in their structure and catalytic properties. At least 4 forms contribute significantly to primary productivity in the ocean. To date, the kinetic isotope effects (KIEs) for RubisCOs, the degree to which the d13C of the C fixed differs from the CO2 substrate used, have only been determined for one form IA RubisCO (chemoautotrophic symbionts of the clam Solemya; 24.5.), two form IB enzymes (spinach and Synechococcus PCC 6301; 29 and 22., respectively) and two form II's (Rhodospirillum rubrum and Riftia pachyptila symbionts; ~18. for both). Determining the KIEs of different RubisCOs is critical to understanding, interpreting, and modeling of d13C values in oceanography and global carbon. The PIs are assessing the KIEs for form IC and ID RubisCOs, to estimate the full range of isotope fractionations by these enzymes. The RubisCO enzymes are being isolated and purified from representative marine phytoplankton and autotrophic bacteria. They are studying form IC RubisCO from the model species Rhodobacter sphaeroides and marine isolate S185-9A1, and form ID RubisCO from the coccolithophore Emiliania huxleyi and the diatom Skeletonema costatum. The isolated proteins are being characterized with respect to amino acid sequence, composition, Vmax, and Km (CO2). Finally, the kinetic isotope effects are being determined by high precision methods.