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

Abstract

Ribulosebisphosphate carboxylase (RuBP carboxylase)forms a stable model complex containing stoichiometric amounts of enzyme sites, activator C0(2), divalent activator cation, and the transition-state analogue carboxyarabinitol bisphosphate (CABP). Incorporation of Mn(2+) in the model complex permits investigation of the environment of the activator cation by electron spin resonance (ESR)techniques. Measurements at 9 GHz on the Mn(2+)-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. In contrast, the spectrum produced from Mn(2+) bound to R. rubrum enzyme exhibits an intense powder pattern for the central fine structure transition; the other four fine structure transitions produce powder patterns that are in homogeneously broadened and therefore are not as apparent.Low-temperature measurements at high field (35 GHz) result in substantially simplified spectra. The spectrum of Mn(2+) bound to the R. rubrum enzyme shows less fine structure than the spectrum of Mn(2+) bound in the octameric spinach enzyme complex, where substantial hyperfine splitting is resolved in three of the five fine structure transitions. Measurements at 35 GHz on Mn (2+) bound in the dimeric R. rubrum enzyme complex produce spectra in which only the central fine structure transition produces a prominent signal. However, these samples are characterized by several narrow spectral features which permit investigation of the identity of Mn(2+)ligands by 170 perturbation techniques. Preparation of the R. rubrum RuBP carboxylase model complex in (17)O-enriched water results in a sample which exhibits an obviously broadened 35-GHz Mn(2+) spectrum in comparison to unenriched samples. Removal of H(2)(17)O by gel filtration abolished the spectral broadening, indicating that the Mn(2+)-coordinated water molecules can slowly exchange. No spectral broadening was detectable due to (17)O in the carbamate oxygens derived from activator C(17)O(2). NMR relaxation rate,measurement sat 24.3 MHz demonstrate that stoichiometric amounts of carboxyarabinitol bisphosphate eliminate enhancement of the proton relaxation rate observed in ternary enzyme-C0(2)-Mn(2+) complexes prepared by using dimeric R. rubrum enzyme. This observation, coupled with results of the H(2)(17)O ESR experiments,is compatible with the suggestion that the water molecules which coordinate directly to bound Mn(2+) are nonexchangeable on an NMR time scale but can be displaced by solvent water within 1-2 h. Carboxyarabinitol bisphosphate was selectively enriched with (17)O in the carboxyl group or in the oxygen on C-2. Mn(2+)-containing complexes prepared with either of the (17)O-enriched analogues produced spectra which were broadened in comparison to matched (l6)O controls. Thus,Mn(2+) coordinates directly to CABP, arguing for the participation of cation in the catalytic process.