CONFORMATIONAL TRANSITIONS OF THE SARCOPLASMIC-RETICULUM CA-ATPASE STUDIED BY TIME-RESOLVED EPR AND QUENCHED-FLOW KINETICS

We have used time-resolved electron paramagnetic resonance (EPR) and quenched-flow kinetics in order to investigate the dynamics of Ca-ATPase conformational changes involved in Ca2+ pumping in sarcoplasmic reticulum (SR) membranes at 2 degrees C. The Ca-ATPase was selectively labeled with an iodoacetamide spin label (IASL), which yields EPR spectra sensitive to enzyme conformational changes during ATP-induced enzymatic cycling. The addition of ATP, AMPPCP, CrATP, or ADP decreased the rotational mobility of a fraction of the probes, indicating a distinct protein conformational state corresponding to this probe population, while P-i under conditions producing ''backdoor'' phosphorylation produced no spectral change. Transient changes in the amplitude of the restricted component associated with the pre-steady state of Ca2+ pumping were detected with 10 ms time resolution after an [ATP] jump produced by laser flash photolysis of caged ATP in the EPR sample. The laser energy was adjusted to generate 100 mu M ATP from 1 mM caged ATP. At 0.1 M KCl, the EPR transient consisted of a brief initial lag phase, a monoexponential phase with a rate of 20 s(-1), and a decay back to the initial intensity after the ATP had been consumed. Raising [KCl] from 0.1 to 0.4 M slowed the rate of the exponential phase from 20 to 6 s(-1). Lowering the pH from 7 to 6, which increased the rate of caged ATP photolysis, eliminated the lag but did not change the apparent rate of the EPR signal rise. Parallel acid quenched-flow experiments conducted at 0.1 M KCl and 100 mu M ATP produced fast (50-58 s(-1)) and slow (20 s(-1)) phases of phosphoenzyme formation. Increasing [KCl] from 0.1 to 0.4 M decreased the rate of the slow phase of phosphorylation from 20 to 5 s(-1), without affecting the fast phase. The close correlation between the slow phase of phosphorylation and the exponential phase of the EPR signal suggests that the spin probe monitors a conformational event associated with phosphoenzyme formation in a population of catalytic sites with delayed kinetics. We propose that this constraint is imposed by conformational coupling between the catalytic subunits in a Ca-ATPase oligomer and that, consequently, the EPR signal reflects changes in quaternary protein structure as well as changes in secondary and tertiary structure associated with ATP-dependent phosphorylation.