Electron spin resonance studies of ribulose bisphosphate carboxylase: identification of activator cation ligands

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Abstract
Ribulosebisphosphate carboxylase (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 ESR. Measurements at 9 GHz on the Mn2+-containing complex prepared by using dimeric Rhodospirillum rubrum enzyme produce a spectrum which indicates that the cation is bound in an anisotropic environment. Measurements at 9 GHz on the spinach enzyme model complex produce a spectrum in which several of the fine structure transitions are obvious. The spectrum produced from Mn2+ bound to R. rubrum enzyme exhibits an intense powder pattern for the central fine structure transition; the other 4 fine structure transitions produce powder patterns that are inhomogeneously broadened and therefore are not as apparent. Low-temperature measurements at high field (35 GHz) result in substantially simplified spectra. The spectrum of Mn2+ bound to the R. rubrum enzyme shows less fine structure than the spectrum of Mn2+ bound in the octameric spinach enzyme complex, where substantial hyperfine splitting is resolved in 3 of the 5 fine structure transitions. Measurements at 35 GHz on Mn2+ bound in the dimeric R. rubrum enzyme complex produce spectra in which only the central fine structure transition produces a prominent signal. These samples are characterized by several narrow spectral features which permit investigation of the identity of Mn2+ ligands by 17O perturbation 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 H217O by gel filtration abolished the spectral broadening, indicating that the Mn2+-coordinated water molecules can slowly exchange. No spectral broadening was detectable due to 17O in the carbamate O2 derived from activator C17O2. NMR relaxation rate measurements at 24.3 MHz demonstrate that stoichiometric amounts of CABP eliminate enhancement of the proton relaxation rate observed in ternary enzyme-CO2-Mn2+ complexes prepared by using dimeric R. rubrum enzyme. This observation, coupled with results of the H217O ESR experiments, is compatible with the suggestion that the water molecules which coordinate directly to bound Mn2+ are nonexchangeable on an NMR time scale but can be displaced by solvent water within 1-2 h. CABP was selectively enriched with 17O in the carboxyl group or in the "O" on C-2. Mn2+-containing complexes prepared with either of the 17O-enriched analogs produced spectra which were broadened in comparison to matched 16O controls. Thus, Mn2+ coordinates directly to CABP, arguing for the participation of cation in the catalytic process.

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