INTRAMOLECULAR CHARGE-TRANSFER IN THE BACTERIORHODOPSIN MUTANTS ASP85-]ASN AND ASP212-]ASN - EFFECTS OF PH AND ANIONS

The photovoltage kinetics of the bacteriorhodopsin mutants Asp212-->As n and Asp85-->Asn after excitation at 580 nm have been investigated in the pH range from 0 to 11. With the mutant Asp85-->Asn (D85N) at pH 7 no net charge translocation is observed and the signal is the same, b oth in the presence of Cl- (150 mM) and in its absence (75 mM SO42-). Under both conditions the color of the pigment is blue (lambda(max) = 615 nm). The time course of the photovoltage kinetics is similar to th at of the acid-blue form of wild-type, except that an additional trans ient charge motion occurs with time constants of 60 mu s and 1.3 ms, i ndicating the transient deprotonation and reprotonation of an unknown group to and from the extracellular side of the membrane. It is sugges ted that this is the group XH, which is responsible for proton release in wild-type, At pH 1, the photovoltage signal of D85N changes upon t he addition of Cl- from that characteristic for the acid-blue state of wild-type to that characteristic for the acid-purple state. Therefore , the protonation of the group at position at 85 is necessary, but not sufficient for the chloride-binding. At pH 11, well above the pK(a) o f the Schiff base, there is a mixture of ''M-like'' and ''N-like'' sta tes. Net proton transport in the same direction as in wild-type is res tored in D85N from this N-like state, With the mutant Asp212-->Asn (D2 12N), time-resolved photovoltage measurements show that in the absence of halide ions the signal is similar to that of the acid-blue form of wild-type and that no net charge translocation occurs in the entire p H range from 0 to 11. Upon addition of Cl- in the pH range from 3.8 to 7.2 the color of the pigment returns to purple and the photovoltage e xperiments indicate that net proton pumping is restored. However, this Cl--induced activation of net charge-transport in D212N is only parti al. Outside this pH range, no net charge transport is observed even in the presence of chloride, and the photovoltage shows the same chlorid e-dependent features as those accompanying the acid-blue to acid-purpl e transition of the wild-type.