Identification of Ligands of the Activator Cation of Ribulose Bisphosphate Carboxylase

Abstract

Ribulose bisphosphate (RuBP) carboxylase forms a stable model complex containing stoichiometric amounts of enzyme sites, activator CO2, divalent activator cation, and the transition state analog, carboxyarabinitol bisphosphate (CABP). Incorporation of Mn2+ in the model complex permits investigation of the environment of the activator cation by electron spin resonance (ESR) techniques. Preparation of the R. rubrum RuBP carboxylase model complex in 17O enriched water results in a sample which exhibits an obviously broadened 35 GHz Mn2+ spectrum in comparison to unenriched samples. Removal of H2 17O by gel filtration abolished the spectral broadening, which is due to superhyperfine coupling, indicating that the Mn2+-coordinated water molecules can slowly exchange. NMR relaxation rate measurements demonstrate that the water molecules which coordinate directly to bound Mn2+ in the CABP-containing complex are non-exchangeable on an NMR time scale. Mn2+ containing complexes prepared with 17O enriched CABP produced spectra which were broadened in comparison to matched 16O controls. Thus, Mn2+ coordinates directly to CABP, arguing for participation of cation in the catalytic process.