Bacteriorhodopsin D85N: three spectroscopic species in equilibrium.
Ground-state absorbance measurements show that BR from Halobacterium halobium containing asparagine at residue 85 (D85N) exists as three distinct chromophoric states in equilibrium. In the pH range 6-12 the absorbance spectra of the three states are demonstrated to be similar to flash-induced spectral intermediates which comprise the latter portion of the wild-type BR photocycle. One of the states absorbs maximally at 405 nm, has a deprotonated Schiff base, and contains predominantly the 13-cis form of retinal, identifying it as a close homologue of the M intermediate in the BR photocycle. The other species possess absorbance maxima with correspondence to those of the wild-type N (570 nm) and O (615 nm) photointermediates. The retinal composition of the O-like form was found to be dominated by all-trans isomer. The pH dependence of the concentrations of the equilibrium species corresponds closely with the pH dependence of the M, N, and O photointermediates. These data support kinetic models which emphasize the role of back-reactions during the photocycle of bacteriorhodopsin. Energetic and spectral characterization of the D85N ground-state equilibrium supports its use as a model for elucidating molecular transitions comprising the latter portion of the BR photocycle.
Turner
G J
GJ
Department of Biochemistry & Biophysics, University of California, San Francisco 94143.
Miercke
L J
LJ
Thorgeirsson
T E
TE
Kliger
D S
DS
Betlach
M C
MC
Stroud
R M
RM
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003624:Darkness; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D019617:Halobacterium salinarum / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008433:Mathematics; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012172:Retinaldehyde / Q000302:isolation & purification
D011994:Recombinant Proteins; D001436:Bacteriorhodopsins; D012172:Retinaldehyde