We measured the M-N transition of wild-type bacteriorhodopsin (pH 9, 10°C) by time-resolved x-ray diffraction study at SPring8 BL45XU-A. We confirmed the accumulation of M and N intermediates by absorbance measurements, and we found that the time resolution of x-ray diffraction experiments (244 ms) was sufficient to resolve the M-N transition. From the x-ray diffraction data, three components were decomposed by singular value decomposition analysis. The existence of three components in the M3 N3 BR reaction revealed that BR changes its structure during the M-N transition. Moreover, the difference Fourier maps of reconstituted fast and slow decay components clearly showed that the electron density distributions of the F helix changes in the M-N transition. The observed structural change at the F helix will increase access of the Schiff base and D96 to the cytoplasmic surface and facilitate the proton transfer steps that begin with the decay of the M state.A n ion pump protein transports ions across the membrane against the chemical potential. A simple idea of the ion pumping mechanism is that the ion-binding states of the ion pump are linked to protein conformations to switch the ion transport pathway from one membrane surface to another surface (1). Bacteriorhodopsin (BR) is one of the proton transport proteins that make use of absorbed photon energy. Its photocycle is characterized by distinct spectra of the metastable intermediate in the visible range as J, K, L, M, N, and O. The most important steps for the proton pump are deprotonation and reprotonation of the Schiff base. The former is the proton transfer from the Schiff base to D85 and the latter is the proton transfer from D96 to the Schiff base. Proton transfer from the Schiff base to D85 occurs in the L-M transition. The structure of the M intermediate was investigated by diffraction methods under stabilizing conditions (2-4). The obtained data showed that the structure of the M intermediate differed largely from the unphotolyzed state. It was shown that this structural change is closely related to the deprotonation of the Schiff base; in the original structure (conformation E) the proton channel is open to the extracellular side, and when an M-type conformation is assumed (conformation C), it is open to the cytoplasmic side (5-7). This structural transition after the first proton transfer prevents reverse proton transfer from D85 to the Schiff base. Thus, the alternating protein conformation model explains the proton transport mechanism (7). The Schiff base is reprotonated from D96 during the M-N transition. Because D96 is located in a hydrophobic environment, it has unusual high pK and it is protonated in an unphotolyzed state. The pK of D96 should be lowered during the M-N transition to transfer a proton. Therefore, an important event in lowering the D96 pK is expected to occur at the M-N transition.Structural studies on photointermediates have been carried out by accumulating specific intermediates using chemical treatments (3, 4), mutations (8, 9),...