Specific effects of chloride on the photocycle of E194Q and E204Q mutants of bacteriorhodopsin as measured by FTIR spectroscopy.
Low-temperature Fourier transform infrared spectroscopy has been used to study mutants of Glu194 and Glu204, two amino acids that are involved in proton release to the extracellular side of bacteriorhodopsin. Difference spectra of films of E194Q, E204Q, E194Q/E204Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q at 243, 277, and 293 K and several pH values were obtained by continuous illumination. A specific effect of Cl(-) ions was found for the mutants, promoting a N-like intermediate at alkaline pH and an O' intermediate at neutral or acid pH. The apparent pK(a) of Asp85 in the M intermediate was found to be decreased for E194Q in the presence of Cl(-) (pK(a) of 7.6), but it was unchanged for E204Q, as compared to wild-type. In the absence of Cl(-) (i.e., in the presence of SO(4)(2)(-)), mutation of Glu194 or of Glu204 produces M- (or M(N), M(G))-like intermediates under all of the conditions examined. The absence of N, O, and O' intermediates suggests a long-range effect of the mutation. Furthermore, it is suggested that Cl(-) acts by reaching the interior of the protein, rather than producing surface effects. The effect of low water content was also examined, in the presence of Cl(-). Similar spectra corresponding to the M(1) intermediate were found for dry samples of both mutants, indicating that the effects of the mutations or of Cl(-) ions are confined to the second part of the photocycle. The water O-H stretching data further confirms altered photocycles and the effect of Cl(-) on the accumulation of the N intermediate.
Lazarova
Tzvetana
T
Unitat de Biofísica, Departament de Bioquímica i de Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra (Cerdanyola del Vallès), Barcelona 08193, Spain.
Sanz
Carolina
C
Sepulcre
Francesc
F
Querol
Enric
E
Padrós
Esteve
E
D019943:Amino Acid Substitution / Q000235:genetics*; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002712:Chlorides / Q000737:chemistry*; D005615:Freezing; D018698:Glutamic Acid / Q000235:genetics* / Q000378:metabolism; D005973:Glutamine / Q000235:genetics* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008027:Light; D016297:Mutagenesis, Site-Directed; D011189:Potassium Chloride / Q000737:chemistry; D011522:Protons; D017550:Spectroscopy, Fourier Transform Infrared; D014867:Water / Q000737:chemistry
D002712:Chlorides; D011522:Protons; D014867:Water; D005973:Glutamine; D018698:Glutamic Acid; D001436:Bacteriorhodopsins; D011189:Potassium Chloride