BACTERIORHODOPSINS INTRAMOLECULAR PROTON-RELEASE PATHWAY CONSISTS OF A HYDROGEN-BONDED NETWORK

In its proton-pumping photocycle, bacteriorhodopsin releases a proton to the extracellular surface at pH 7 in the transition from intermedia te L to intermediate M. The proton-release group, named XH, was assign ed in low-temperature FT-IR studies to a single residue, E204 [Brown, L. S., Sasaki, J., Kandori, H., Maeda, A., Needleman, R., and Lanyi, J . K. (1995) J. Biol. Chem. 270, 27122-27126]. The time-resolved room-t emperature step-scan FT-IR photocycle studies on wild-type and E204Q-, and E204D-mutated bacteriorhodopsin, which we present here, show in c ontrast that the FT-IR data give no evidence for deprotonation of E204 in the L-to-M transition. Therefore, it is unlikely that E204 represe nts XH. On the other hand, IR continuum absorbance changes indicate in tramolecular proton transfer via an H-bonded network to the surface of the protein. It appears that this H-bonded network is spanned between the Schiff base and the protein surface. The network consists at leas t partly of internally bound water molecules and is stabilized by E204 and R82. Other not yet identified groups may also contribute. At pH 5 , the intramolecular proton transfer to the surface of the protein see ms not to be disturbed. The proton seems to be buffered at the surface and later in the photocycle released into the bulk during BR recovery . Intramolecular proton transfer via a complex H-bonded network is pro posed to be a general feature of proton transfer in proteins.