COVALENTLY FOUND PH-INDICATOR DYES AT SELECTED EXTRACELLULAR OR CYTOPLASMIC SITES IN BACTERIORHODOPSIN .2. ROTATIONAL ORIENTATION OF HELIX-D AND HELIX-E AND KINETIC CORRELATION BETWEEN M-FORMATION AND PROTON RELEASE IN BACTERIORHODOPSIN MICELLES

The kinetics of the light-induced proton release in bacteriorhodopsin/ lipid/detergent micelles was monitored with the optical pH-indicator f luorescein bound covalently to positions 127-134 (helices D and E and the DE loop) on the extracellular side of the protein (the proton rele ase side). Single cysteine residues were introduced in these positions by site-directed mutagenesis, and fluorescein was attached to the sul fhydryl group by reaction with (iodoacetamido)fluorescein. Two charact eristic proton release times (approximately 20 and 70 mu s) were obser ved. The faster time constant was recorded when fluorescein was attach ed to positions 127, 130, 131, 132, and 134, while the slower time was observed with the indicator bound to positions 128, 129, and 133. The results are rationalized by assuming specific helical wheel orientati ons for helics D and E and by making a choice for the residues in the DE loop: (i) The fast time constants occur with fluorescein either att ached to residues 130, 131, and 132 that form the DE loop or when poin ting toward the interior of the protein with its aqueous proton channe l [residues 127 (helix D) and 134 (helix E)]. (ii) The slower time con stants are detected with fluorescein exposed to the exterior lipid/det ergent phase when bound to residues 128, 129 (both helix D), and 133 ( helix E). This interpretation is supported by measurements of the pola rity of the label environment which indicate for fluorescein in group i a more hydrophilic environment and for group ii a more hydrophobic e nvironment. The fastest proton release time (10 mu s) was observed wit h fluorescein bound to position 127. This release time equals the rise time of the main component in the formation of the M intermediate. Fo r positions 130, 131, 132, and 134, there is an apparent delay of at l east 10 mu s between these two processes. The activation energy of the fast proton release time: for position 130 on the extracellular side (52 +/- 5 kJ/mol) is similar to that of the slowest component in the r ise of the M intermediate (65 +/- 5 kJ/mol). These results suggest tha t the formation of M and the proton release are kinetically coupled.