The photocycle, electrical charge translocation, and release and uptake of protons from the aqueous phase were investigated for bacteriorhodopsin mutants with aspartic acid-96 replaced by asparagine or glutamic acid. At

Electrogenic and redox events in the reaction-centre complexes from Rhodopseudomonas viridis have been studied. In contrast to the previous points of view it is shown that all the four hemes of the tightly bound cytochrome c have different Em values (-60, + 20, + 310 and + 380 mV). The first three hemes reveal ci absorption maxima at 554 nm, 552 nm and 556 nm respectively. The 380-mV heme displays a split ci band with a maximum at 559 nm and a shoulder at 552 nm. Such a splitting is due to non-degenerated Qx and Q, transitions in the ironporphyrin ring as demonstrated by magnetic circular dichroism spectra. Fast kinetic measurements show that, at redox potentials when only high-potential hemes c-559 and c-556 are reduced, heme c-559 appears to be the electron donor to P-960' (z = 0.32 ps) whereas heme c-556 serves to rereduce c-559 (z = 2.5 ps). Upon reduction of the third heme (c-552), the P-960' reduction rate increases twofold (z = 0.17 ps) and all photoinduced redox events within the cytochrome appear to be complete in less than 1 ps after the flash. The following sequence of the redox centers is tentatively suggested: c-554, c-556, c-552, c-559, P-960.To study electrogenesis, the reaction-centre complexes from Rps. viridis were incorporated into asolectin liposomes, and fast kinetics of laser flash-induced electric potential difference has been measured in proteoliposomes adsorbed on a phospholipid-impregnated film. The electrical difference induced by a single 15-11s flash was found to be as high as 100 mV. The photoelectric response has been found to involve four electrogenic stages associated with (I) QA reduction by P-960; (11) reduction of P-960+ by heme c-559; (111) reduction of c-559 by c-556 and (IV) protonation of Qg-. The relative contributions of stages I, 11,111 and IV are found to be equal to 70%, 15%, 5% and lo%, respectively, of the overall electrogenic process. At the same time, the first three respective distances along the axis normal to the membrane plane covered by electrons, calculated from X-ray data of Deisenhofer et al. [J, Mol. Biol. 180, 385-398 (1984)], are 22%, 18.5% and 26%. This indicates that the efficiency of electrogenic phases depends first of all upon the value of the dielectric constant of the respective membrane regions rather than upon the distance between the redox groups involved. The efficiency is higher, the deeper these groups are immersed in the membrane.The concept of transmembrane electron flow in the coupling sites of redox chains was put forward by Mitchell virtually simultaneously with the general chemiosmotic principle of energy coupling via protonmotive force [l]. Whereas the general formulation of the chemiosmotic theory proved to be accepted by the majority of bioenergeticists, there is still considerable debate on the electrogenic transmembrane electron transfer as a mechanism of ApH generation.There are many indications that electron transfer across the coupling membrane does indeed occur in reaction centre complexes of photosynthetic bacteria (reviewed...

The kinetics of 3 photoinduced responses of bacteriorhodopsin have been compared: spectral changes. pH shifts in a suspension of open purple membrane sheets and electric potential generation by the sheets incorporated into a lipid-impregnated collodion film. In the presence of a pH-buffer, the H+ release by bacteriorhodopsin was shown to correlate with the formation of the M412 intermediate and the microsecond phase of the potential generation. The H+/M412 ratio is equal to 0.7kO.l if the ionic strength of the solution is high. In the absence of the buffer, the H+ release proved to be much slower than spectra1 and electric responses. The kinetics of H+ uptake by bacteriorhodopsin is close to M412 decay and to the electrogeneous millisecond phase in both the presence and absence of the pH buffer. The bacteriorhodopsin-induced proton release phase accounts for about 20%, and the uptake phase for about 80% of the overall potential. This is compatible with the model assuming that the proton start-out point -possibly, the protonated Schiff base connecting lysine 216 with retina1 -is closer to the outer rather than the inner (cytoplasmic) surface of the bacteria1 membrane. Bacteriorhodopsin Retinal location pH indicator

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