Fast stages of photoelectric processes in biological membranes. I. Bacteriorhodopsin

Bacteriorhodopsin-containing fragments of Halobacterium halobium membrane (bacteriorhodopsin sheets) were incorporated into a lecithin-impregnated collodion film, and fast stages of flash-induced electrogenesis were measured by two electrodes separated by this film. It is found that a single turnover of bacteriorhodopsin results in an electrogenic response composed of three main stages of the following tau: the first less than 200 ns, the second 15 - 70 microseconds and the third 10 ms. The second and third phases are of the same direction as an electric response to continuous illumination, whereas the first one is oppositely directed. The microseconds and ms stages were shown to correlate, in the first approximation, with formation and decomposition of the bacteriorhodopsin intermediate absorbing with 412 nm, respectively. Both the second and third phases of the photoelectric response are sums of at least two exponents. The third stage is specifically inhibition by La3+ ions which are also shown to decrease the rate of regeneration of the original bacteriorhodopsin absorbing at 570 nm from the intermediate absorbing at 412 nm. Acidification of the medium induces parallel inhibition of the second and third phases and of formation of the intermediate absorbing at 412 nm as if protonation of a group with pK = 3.6 were responsible for this inhibition. The first (opposite) phase survives acidification. It even increases at pH lower than 1.5. At such a low pH, one can show a good correlation of decays of photopotential and of a bacteriorhodopsin bathointermediate. The decays are biphasic (tau 1 = 200 microseconds and tau 2 = 2 ms), formation of both the photopotential and the bathointermediate being faster than 200 ns. At higher pH, when a three-phase photoelectric response is revealed, decay of the formed electric potential difference gives the average tau value of about 1 s. It can be accelerated by compounds that increase ionic conductance of biomembranes. At pH below 4, fluoride is found to completely inhibit the second and third phases, so that only the first phase is observed. The results are discussed in terms of a scheme postulating that the first electrogenic phase is a result of translocation of the protonated Schiff base inside the membrane due to a light-induced conformation change in retinal or protein. The second and third phases are explained by H+ transfer from the Schiff base to the outer membrane surface and from inner (cytoplasmic) surface of membrane to the Schiff base, respectively.