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 SURFACE TO BULK

The kinetics of the light-induced release and uptake of protons was monitored with the optical pH-indicator fluorescein covalently bound to various sites on the extracellular and cytoplasmic surfaces of bacteriorhodopsin. Selective labeling was achieved by reacting (iodoacetamido)fluorescein with the single cysteine residues in bacteriorhodopsin introduced at the desired positions by site-directed mutagenesis. All measurements were performed with bacteriorhodopsin micelles in phospholipid/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 bacteriorhodopsin mutants with cysteine residues on the cytoplasmic side. The proton release time detected with fluorescein attached to the extracellular surface (the proton release side) at position 72 (in the loop connecting 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 mutants labeled at positions 35, 101, 160, 229, and 231 in the cytoplasmic loop region (the proton uptake side), the released proton was observed with a time of 61 +/- 4 mu s. This was about 3-fold slower than the release time on the extracellular side, but still significantly faster than that measured with pyranine in the bulk phase. These results suggest 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 experiments in which the proton mobility along the micellar surface was varied by adding phospholipids with headgroups of different pK(a)'s to the bacteriorhodopsin/CHAPS micelles. With the label on the cytoplasmic side, the amplitude of the Light-induced transient protonation change increased or decreased, respectively, when DMPC (pK 2.2; proton dwell time approximate to 10 ns) or DMPA (pK 8.0; proton dwell time approximate to 10 ms) was added. However, no effect of the phospholipids was detected with the indicator on the extracellular side. These observations of faster proton diffusion along the micellar surface than their equilibration from the surface to the bulk support bioenergetic models of efficient proton coupling along the membrane surface between proton 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 correlate it with a specific photocycle intermediate.