Crystallographic structure of the retinal and the protein after deprotonation of the Schiff base: The switch in the bacteriorhodopsin photocycle

We illuminated bacteriorhodopsin crystals at 210 K to produce, in a photo-stationary state with 60% occupancy, the earliest M intermediate (M,) of the photocycle. The crystal structure of this state was then determined from X-ray diffraction to 1.43 Angstrom resolution. When the refined model is placed after the recently determined structure for the K intermediate but before the reported structures for two later M states, a sequence of structural changes becomes evident in which movements of protein atoms and bound water are coordinated with relaxation of the initially strained photoisomerized 13-cis,15-anti retinal. In the K state only retinal atoms are displaced, but in M, water 402 moves also, nearly 1 Angstrom away from the unprotonated retinal Schiff base nitrogen. This breaks the hydrogen bond that bridges them, and initiates rearrangements of the hydrogen-bonded network of the extracellular region that develop more fully in the intermediates that follow. In the M-1 to M-2 transition, relaxation of the C-14-C-15 and Q,5 NZ torsion angles to near 180degrees reorients the retinylidene nitrogen atom from the extracellular to the cytoplasmic direction, water 402 becomes undetectable, and the side-chain of Arg82 is displaced strongly toward Glu194 and Glu204. Finally, in the M-2 to M-2' transition, correlated with release of a proton to the extracellular surface, the retinal assumes a virtually fully relaxed bent shape, and the 13-methyl group thrusts against the indole ring of Trp182 which tilts in the cytoplasmic direction. Comparison of the structures of M, and M2 reveals the principal switch in the photocycle: the change of the angle of the C-15=NZ-CE plane breaks the connection of the unprotonated Schiff base to the extracellular side and establishes its connection to the cytoplasmic side. (C) 2002 Elsevier Science Ltd. All rights reserved.