CONFORMATIONAL FLEXIBILITY OF ARGININE-82 AS SOURCE FOR THE HETEROGENEOUS AND PH-DEPENDENT KINETICS OF THE PRIMARY PROTON-TRANSFER STEP IN THE BACTERIORHODOPSIN PHOTOCYCLE - AN ELECTROSTATIC MODEL

We use equilibrium thermodynamic concepts to relate protein conformati onal and protonation substates and their pH-dependent population to ki netic schemes for the rise of the M intermediate in the photocycle of bacteriorhodopsin. Conformational flexibility of arginine R82 is descr ibed by a two-state model, The analysis accounts for the electrostatic coupling between its orientation and hydrogen ion titration and prese nts a structural basis for the linkage between the protonation states of the primary proton acceptor, aspartate D85, and the extracellular r elease group, glutamate E204. We find that the charge state of D85 is a significant determinant for the orientation of R82. The molecular mo del predicts the following: the primary proton transfer to D85 can be described by a kinetic scheme with two heterogeneous substates, They c ontrol the event with different activation parameters due to the reori entation of R82 away from the chromophore binding site. Their populati on depends on the external pH and the proton exchange equilibrium betw een the membrane buried residues and the bulk aqueous solvent. Proton transfer in the physiologic pH range is strongly activated and followe d by the reorientation of R82 which shifts the equilibrium toward comp lete transfer. In the alkaline pH region a different mechanism operate s, which involves the increased population of a substate with already reoriented R82 as a consequence of the deprotonation of E204, leading to accelerated proton transfer. Assuming full proton exchange equilibr ium with the bulk water on the millisecond time scale leads to an incr eased population of substates which are non-productive for proton tran sfer.