STRUCTURE OF THE N INTERMEDIATE OF BACTERIORHODOPSIN REVEALED BY X-RAY-DIFFRACTION

X-ray diffraction experiments revealed the structure of the N photoint ermediate of bacteriorhodopsin, Since the retinal Schiff base is repro tonated from Asp-96 during the M to N transition in the photocycle, an d Asp-96 is reprotonated during the lifetime of the N intermediate, or immediately after, N is a key intermediate for understanding the ligh t driven proton pump. The N intermediate accumulates in large amounts during continuous illumination of the F171C mutant at pH 7 and 5 degre es C. Small but significant changes of the structure were detected in the x-ray diffraction profile under these conditions. The changes were reversible and reproducible, The difference Fourier map indicates tha t the major change occurs near helix F. The observed diffraction chang es between N and the original state were essentially identical to the diffraction changes reported for the M intermediate of the D96N mutant of bacteriorhodopsin, Thus, we find that the protein conformations of the M and N intermediates of the photocycle are essentially the same, in spite of the fact that in M the Schiff base is unprotonated and in N it is protonated, The observed structural change near helix F will increase access of the Schiff base and Asp-96 to the cytoplasmic surfa ce and facilitate the proton transfer events that begin with the decay of the M state.