COVALENTLY BOUND 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 fluorescein bound covalently to positions 127-134 (helices D and E and the DE loop) on the extracellular side of the protein (the proton release side). Single cysteine residues were introduced in these positions by site-directed mutagenesis, and fluorescein was attached to the sulfhydryl group by reaction with (iodoacetamido)fluorescein. Two characteristic proton release times (approximately 20 and 70 mu s) were observed. The faster time constant was recorded when fluorescein was attached 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 orientations 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 attached to residues 130, 131, and 132 that form the DE loop or when pointing toward the interior of the protein with its aqueous proton channel [residues 127 (helix D) and 134 (helix E)]. (ii) The slower time constants are detected with fluorescein exposed to the exterior lipid/detergent phase when bound to residues 128, 129 (both helix D), and 133 (helix E). This interpretation is supported by measurements of the polarity of the label environment which indicate for fluorescein in group i a more hydrophilic environment and for group ii a more hydrophobic environment. The fastest proton release time (10 mu s) was observed with fluorescein bound to position 127. This release time equals the rise time of the main component in the formation of the M intermediate. For positions 130, 131, 132, and 134, there is an apparent delay of at least 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 rise of the M intermediate (65 +/- 5 kJ/mol). These results suggest that the formation of M and the proton release are kinetically coupled.