COVALENTLY BOUND PH-INDICATOR DYES AT SELECTED EXTRACELLULAR OR CYTOPLASMIC SITES IN BACTERIORHODOPSIN .1. PROTON MIGRATION ALONG THE SURFACE OF BACTERIORHODOPSIN MICELLES AND ITS DELAYED TRANSFER FROM SURFACETO BULK

The kinetics of the light-induced release and uptake of protons was mo nitored with the optical pH-indicator fluorescein covalently bound to various sites on the extracellular and cytoplasmic surfaces of bacteri orhodopsin. Selective labeling was achieved by reacting (iodoacetamido )fluorescein with the single cysteine residues in bacteriorhodopsin in troduced at the desired positions by site-directed mutagenesis. All me asurements were performed with bacteriorhodopsin micelles in phospholi pid/detergent mixtures in 150 mM. KCl at 22 degrees C, pH 7.3. Neither the replacements by cysteine nor the subsequent labeling affected the absorption spectrum of bacteriorhodopsin and the rise times of the M intermediate. Only the decay of M was altered for some bacteriorhodops in mutants with cysteine residues on the cytoplasmic side. The proton release time detected with fluorescein attached to the extracellular s urface (the proton release side) at position 72 (in the loop connectin g helices B and C) or 130 (DE loop) was 22 +/- 4 mu s, clearly faster than that measured with pyranine in the aqueous bulk phase (125 +/- 10 mu s for wild-type and all mutants studied). For bacteriorhodopsin mu tants labeled at positions 35, 101, 160, 229, and 231 in the cytoplasm ic loop region (the proton uptake side), the released proton was obser ved with a time of 61 +/- 4 mu s. This was about 3-fold slower than th e release time on the extracellular side, but still significantly fast er than that measured with pyranine in the bulk phase. These results s uggest that the released protons are retained on the micellar surface and move more rapidly along this surface to the cytoplasmic side than from the surface to the bulk medium. This conclusion is supported by e xperiments in which the proton mobility along the micellar surface was varied by adding phospholipids with headgroups of different pK(a)'s t o the bacteriorhodopsin/CHAPS micelles. With the label on the cytoplas mic side, the amplitude of the Light-induced transient protonation cha nge increased or decreased, respectively, when DMPC (pK 2.2; proton dw ell time approximate to 10 ns) or DMPA (pK 8.0; proton dwell time appr oximate to 10 ms) was added. However, no effect of the phospholipids w as detected with the indicator on the extracellular side. These observ ations of faster proton diffusion along the micellar surface than thei r equilibration from the surface to the bulk support bioenergetic mode ls of efficient proton coupling along the membrane surface between pro ton sources and sinks. Our data also demonstrate that a probe bound to the extracellular (proton release) surface is required to detect the actual appearance of the pumped proton on the protein surface and to c orrelate it with a specific photocycle intermediate.