Characterization of the azide-dependent bacteriorhodopsin-like photocycle of salinarum halorhodopsin.
The photocycle of salinarum halorhodopsin was investigated in the presence of azide. The azide binds to the halorhodopsin with 150 mM binding constant in the absence of chloride and with 250 mM binding constant in the presence of 1 M chloride. We demonstrate that the azide-binding site is different from that of chloride, and the influence of chloride on the binding constant is indirect. The analysis of the absorption kinetic signals indicates the existence of two parallel photocycles. One belongs to the 13-cis retinal containing protein and contains a single red shifted intermediate. The other photocycle, of the all-trans retinal containing halorhodopsin, resembles the cycle of bacteriorhodopsin and contains a long-living M intermediate. With time-resolved spectroscopy, the spectra of intermediates were determined. Intermediates L, N, and O were not detected. The multiexponential rise and decay of the M intermediate could be explained by the introduction of the "spectrally silent" intermediates M1, M2, and HR', HR, respectively. The electric signal measurements revealed the existence of a component equivalent with a proton motion toward the extracellular side of the membrane, which appears during the M1 to M2 transition. The differences between the azide-dependent photocycle of salinarum halorhodopsin and pharaonis halorhodopsin are discussed.
Lakatos
Melinda
M
Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, Szeged, H-6701, Hungary.
Groma
Géza I
GI
Ganea
Constanta
C
Lanyi
Janos K
JK
Váró
György
G
D000042:Absorption; D001386:Azides / Q000494:pharmacology*; D001436:Bacteriorhodopsins / Q000378:metabolism*; D002713:Chlorine / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019617:Halobacterium salinarum / Q000378:metabolism*; D025602:Halorhodopsins / Q000378:metabolism*; D007700:Kinetics; D008027:Light; D008956:Models, Chemical; D011485:Protein Binding; D011522:Protons; D012965:Sodium Chloride / Q000494:pharmacology; D013053:Spectrophotometry; D013431:Sulfates / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors
D001386:Azides; D025602:Halorhodopsins; D011522:Protons; D013431:Sulfates; C012036:sodium sulfate; D012965:Sodium Chloride; D002713:Chlorine; D001436:Bacteriorhodopsins