Structural changes due to the deprotonation of the proton release group in the M-photointermediate of bacteriorhodopsin as revealed by time-resolved FTIR spectroscopy.
One of the steps in the proton pumping cycle of bacteriorhodopsin (BR) is the release of a proton from the proton-release group (PRG) on the extracellular side of the Schiff base. This proton release takes place shortly after deprotonation of the Schiff base (L-to-M transition) and results in an increase in the pKa of Asp85, which is a crucial mechanistic step for one-way proton transfer for the entire photocycle. Deprotonation of the PRG can also be brought about without photoactivation, by raising the pH of the enzyme (pKa of PRG; approximately 9). Thus, comparison of the FTIR difference spectrum for formation of the M intermediate (M minus initial unphotolyzed BR state) at pH 7 to the corresponding spectrum generated at pH 10 may reveal structural changes specifically associated with deprotonation of the PRG. Vibrational bands of BR that change upon M formation are distributed across a broad region between 2120 and 1685 cm(-1). This broad band is made up of two parts. The band above 1780 cm(-1), which is insensitive to C15-deuteration of the retinal, may be due to a proton delocalized in the PRG. The band between 1725 and 1685 cm(-1), on the lower frequency side of the broad band, is sensitive to C15-deuteration. This band may arise from transition dipole coupling of the vibrations of backbone carbonyl groups in helix G with the side chain of Tyr57 and with the C15H of the Schiff base. In M, these broad bands are abolished, and the 3657 cm(-1) band, which is due to the disruption of the hydrogen bonding of a water molecule, probably with Arg82, appears. Loss of the interaction of the backbone carbonyl groups in helix G with Tyr57 and the Schiff base, and separation of Tyr57 from Arg82, may be causes of these spectral changes, leading to the stabilization of the protonated Asp85 in M.
Department of Biology, Center for Biotechnology and Interdisciplinary Studies, Room 2137, Rensselaer Polytechnic Institute, 110 Eighth Street, Troy, New York 12180, USA.
Joel E
JE
Morgan
Ahmet S
AS
Vakkasoglu
Johan
J
Lugtenburg
Robert B
RB
Gennis
Akio
A
Maeda
D001224:Aspartic Acid / Q000737:chemistry; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000528:radiation effects; D002250:Carbon Radioisotopes; D003903:Deuterium; D019617:Halobacterium salinarum / Q000737:chemistry / Q000528:radiation effects; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D010777:Photochemistry; D017433:Protein Structure, Secondary; D011522:Protons; D012545:Schiff Bases / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D014443:Tyrosine / Q000737:chemistry
D002250:Carbon Radioisotopes; D011522:Protons; D012545:Schiff Bases; D001224:Aspartic Acid; D014443:Tyrosine; D001436:Bacteriorhodopsins; D003903:Deuterium