MODIFICATION OF CARBOXYL GROUPS IN BACTERIORHODOPSIN - CHEMICAL EVIDENCE FOR THE INVOLVEMENT OF ASPARTIC-ACID RESIDUES IN THE STRUCTURE AND FUNCTION OF BACTERIORHODOPSIN

Abstract

Covalent modifications of the carboxyl residues of bacteriorhodopsin with alpha-diazo-p-nitroacetophenone (1) under different conditions have been performed. The modified proteins have been characterized for their absorption, photochemical and proton pump activities. A partial characterization in terms of modification site has also been carried out. Three carboxyl residues of dark-adapted bacteriorhodopsin undergo reaction with 1 at pH 4.0, and the resulting protein exhibits absorption and proton pump activity similar to that of the native protein. Dark-adapted bacteriorhodopsin does not react with 1 at pH greater than 6.1. More than one carboxyl residues are modified when light-adapted bacteriorhodopsin is treated with 1 at acidic pH of 4.0 and 5.4. However, near physiological pH (7.2) only one carboxyl residue of light-adapted bacteriorhodopsin reacts with 1. These proteins exhibit absorption bands at 571 nm, fail to show proton translocation, and, upon flash photolysis, exhibit generation of 'M'-like intermediates with tau1/3 of 13.47 ms, and lambda(max) of 400 nm. The modified carboxyl residue is found to be located in the CNBr-9 (residues 72-118) fragment. Reaction of bacteriorhodopsin with 1 at -30-degrees-C under photolytic (lambda greater-than-or-equal-to 500 nm) conditions at pH 7.2 results in the modification of two carboxyl residues, Asp-212 and another one in the CNBr-9 fragment. Such a modified protein exhibits drastically blue-shifted absorption at 400 nm, and does not show any proton translocation or flash photolytic activities. It has been concluded that during the photocycle at least two carboxyl residues exist predominantly in a protonated form. A molecular mechanism for the photocycle is also presented.