Proton transfer from the bulk to the bound ubiquinone Q(B) of the reactioncenter in chromatophores of Rhodobacter sphaeroides: Retarded conveyance by neutral water

The mechanism of proton transfer from the bulk into the membrane protein in terior was studied. The light-induced reduction of a bound ubiquinone molec ule Q(B) by the photosynthetic reaction center is accompanied by proton tra pping. We used kinetic spectroscopy to measure (i) the electron transfer to Q(B) (at 450 nm), (ii) the electrogenic proton delivery from the surface t o the QB site (by electrochromic carotenoid response at 524 nm), and (iii) the disappearance of protons from the bulk solution (by pH indicators). The electron transfer to Q(B)(-) and the proton-related electrogenesis proceed ed with the same time constant of approximate to 100 mu s (at pH 6.2), wher eas the alkalinization in the bulk was distinctly delayed (tau approximate to 400 mu s). We investigated the latter reaction as a function of the pH i ndicator concentration, the added pH buffers, and the temperature. The resu lts led us to the following conclusions: (4 proton transfer from the surfac e-located acidic groups into the Q(B) site followed the reduction of Q(B) w ithout measurable delay; (ii) the reprotonation of these surface groups by pH indicators and hydronium ions was impeded, supposedly, because of their slow diffusion in the surface water layer; and (iii) as a result, the proto ns were slowly donated by neutral water to refill the proton vacancies at t he surface. It is conceivable that the same mechanism accounts for the dela yed relaxation of the surface pH changes into the bulk observed previously with bacteriorhodopsin membranes and thylakoids. Concerning the coupling be tween proton pumps in bioenergetic membranes, our results imply a tendency for the transient confinement of protons at the membrane surface.