Light-induced hydrolysis and rebinding of nonisomerizable bacteriorhodopsin pigment.
Bacteriorhodopsin (bR) is characterized by a retinal-protein protonated Schiff base covalent bond, which is stable for light absorption. We have revealed a light-induced protonated Schiff base hydrolysis reaction in a 13-cis locked bR pigment (bR5.13; lambda(max) = 550 nm) in which isomerization around the critical C13==C14 double bond is prevented by a rigid ring structure. The photohydrolysis reaction takes place without isomerization around any of the double bonds along the polyene chain and is indicative of protein conformational alterations probably due to light-induced polarization of the retinal chromophore. Two photointermediates are formed during the hydrolysis reaction, H450 (lambda(max) = 450 nm) and H430 (lambda(max) = 430 nm), which are characterized by a 13-cis configuration as analyzed by high-performance liquid chromatography. Upon blue light irradiation after the hydrolysis reaction, these intermediates rebind to the apomembrane to reform bR5.13. Irradiation of the H450 intermediate forms the original pigment, whereas irradiation of H430 at neutral pH results in a red shifted species (P580), which thermally decays back to bR5.13. Electron paramagnetic resonance (EPR) spectroscopy indicates that the cytoplasmic side of bR5.13 resembles the conformation of the N photointermediate of native bR. Furthermore, using osmotically active solutes, we have observed that the hydrolysis rate is dependent on water activity on the cytoplasmic side. Finally, we suggest that the hydrolysis reaction proceeds via the reversed pathway of the binding process and allows trapping a new intermediate, which is not accumulated in the binding process.
Aharoni
Amir
A
Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot 76100, Israel.
Ottolenghi
Michael
M
Sheves
Mordechai
M
D001059:Apoproteins / Q000737:chemistry / Q000528:radiation effects; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000528:radiation effects; D004224:Diterpenes; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007536:Isomerism; D007700:Kinetics; D008027:Light; D008958:Models, Molecular; D015394:Molecular Structure; D011485:Protein Binding; D012172:Retinaldehyde / Q000737:chemistry*; D012545:Schiff Bases; D013053:Spectrophotometry
D001059:Apoproteins; D004224:Diterpenes; D012545:Schiff Bases; C031338:13-cis-retinal; D001436:Bacteriorhodopsins; D012172:Retinaldehyde