[Study of intermediate N using mutant forms of bacteriorhodopsin at Asp-96].
It has been found that the N(P, R)-type intermediate of the photocycle is formed in the Asp-96-->Asn mutant at acidic pH. Azide, which strongly activates the M decay in this mutant, allows the N intermediate to be shown also at neutral pH. Under these conditions mutant N decays in a pH-independent fashion. In the presence of azide, the H+ uptake by Asp-96-->Asn mutant bacteriorhodopsin follows the M decay, whereas the N decay occurs at a much slower rate. Two electrogenic stages have been shown to be associated with the M--->bR step in the Asp-96--->Asn mutant photocycle. The faster and slower stages correlate with the M--->N and N--->bR transitions, respectively. In the Asp-96--->Asn mutant, high concentrations of azide are found to increase the M decay rate up to the values higher than those in the wild-type protein, both with or without azide. Such an effect is absent for the Asp-96-->Glu mutant. The activation energies for M--->N and N--->bR transitions in the wild-type protein are equal to 18 and 19 kcal x mole-1, respectively. In the Asp-96-->Asn mutant without azide, the activation energy of the M decay is only 5 kcal x mole-1, whereas in the presence of azide in this mutant the activation energies for M and N decays are 8 and 9 kcal x mole-1, respectively. A scheme of events accompanying the Schiff base reprotonation during the photocycle is discussed.
Danshina
S V
SV
Drachev
L A
LA
Kaulen
A D
AD
Korana
Kh G
KhG
Marti
T
T
Mogi
T
T
Skulachev
V I
VI
D001224:Aspartic Acid / Q000235:genetics*; D001436:Bacteriorhodopsins / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009154:Mutation; D011522:Protons
D011522:Protons; D001224:Aspartic Acid; D001436:Bacteriorhodopsins
Issledovanie intermediatea N s pomoshch'iu mutantnykh form bakteriorhodopsina po Asp-96.