EFFECTS OF HYDROSTATIC-PRESSURE ON THE KINETICS REVEAL A VOLUME INCREASE DURING THE BACTERIORHODOPSIN PHOTOCYCLE

A protein structural change in the photocycle of the proton pump, bact eriorhodopsin, detected earlier in the M photointermediate by diffract ion, consists mainly of changes at the cytoplasmic surface that includ e an outward tilt of the cytoplasmic end of helix F. Such a conformati onal rearrangement would result in greater exposure of the interhelica l cavity to the medium, increased binding of water, and thus an increa se in volume. In order to correlate the structural change with the kin etics of the photoreaction cycle, we measured the effects of hydrostat ic pressure between 1 bar and 1 kbar on the rate constants of the phot ocycles of wild type bacteriorhodopsin and the D96N mutant. Combining the results provided all of the activation volumes and, therefore, the changes of volume in the various states after the K photointermediate is formed. There is an approximately 32 mL/mol volume increase after deprotonation of the retinal Schiff base to the extracellular side, du ring the M(1) --> M(2) reaction, that is not reversed until well after its reprotonation from the cytoplasmic side. The magnitude of this vo lume increase is about as predicted by the increase of the lattice con stant in the M state. It occurs in the photocycle at the proposed repr otonation switch, supporting the idea that this conformation change is what alters the accessibility of the Schiff base from one membrane si de to the other. Additionally, we observe a large positive (approximat ely 50 mL/mol) activation volume for proton exchange between D96 and t he Schiff base of the wild type protein. This is consistent with a req uirement for increased hydration of the protein near D96 for the proto n exchange to occur, as suggested earlier from the specific inhibition of this step by osmotically active solutes.