On the photocycle of 4-ketobacteriorhodopsin.

The artificial pigment 4-ketobacteriorhodopsin is an interesting analog of bacteriorhodopsin. Arguments concerning the scheme of the photocycle of 4-ketobacteriorhodopsin are discussed.

Bacteriorhodopsin. Correspondence of the photocycle and electrogenesis with sites of the molecule.

Correspondence of phases of electrogenesis, photocycle transitions, and proton transfer with the proton transporting groups of bacteriorhodopsin was studied. The structure of bacteriorhodopsin was considered by the file 1c3w and projections of sites of the proton movement pathway onto the normal to the purple membrane were measured. The dielectric permeability of the terminal site of the semichannel Schiff base --> external surface of the purple membrane was noticeably higher than in the center of the membrane.

Photoelectrochemical cycle of bacteriorhodopsin.

The scheme of the bacteriorhodopsin photocycle associated with a transmembrane proton transfer and electrogenesis is considered. The role of conformational changes in the polypeptide chain during the proton transport is discussed.

Synthesis and properties of bacteriorhodopsin analogs containing electron-density labels in the chromophore moiety.

A method for synthesis of retinal analogs labeled with electron-density groups is suggested. The interaction of these polyene compounds with bacterioopsin in apomembrane of Halobacterium salinarum was tested. A retinal analog containing a crown-ether receptor group is able to interact readily with bacterioopsin giving rise to rapid formation of a pigment with absorption maximum at 460 nm. This pigment is capable of undergoing cyclic photoconversion. The crown-bacteriorhodopsin photocycle is extremely slow and its quantum efficiency is very low (approximately 3% of that in native bacteriorhodopsin). This photocycle includes an M-like intermediate with a differential absorption maximum at 380 nm. A retinal analog in which the beta-ionone ring is replaced by ferrocene moiety forms a stable chromoprotein with the main absorption band at 483 nm and a shoulder near 590-610 nm.

13-Demethylbacteriorhodopsin: formation and some properties.

Incorporation of 9-cis-13-demethylretinal into bacterioopsin was shown to form the covalent purple complex. This result was predicted by our hypothesis about the structure of the BR chromophore cavity (Mol. Biologiya 29:1398-1407 (1995) (in Russian)). It supports the hypothesis and eliminates the main objection known from the literature.

C(13)-substituted bacteriorhodopsin analogs.

13-Ethyl-, 13-isopropyl-, 13-tert-butyl-, 13-phenyl-, 13-alpha-naphthyl-, and 13-demethyl-retinals were synthesized and incubated with bacterioopsin (bO) to give the corresponding bacteriorhodopsin (bR) analogs. The capability of the 13-tert-butyl- and 13-alpha-naphthyl-bRs to exist and to photocycle shows that apparently around C(13) of the chromophore there lies a large enough cavity. A study of the light-induced conversions of the artificial pigments prepared has shown that the introduction at position 13 of the chromophore of the hydrocarbon substituents bulkier than that of the natural bR diminished the amplitudes of the electric photoresponses. Bulky C(13)-substituents or absence of substitution at that position decelerated the relaxation of the M-intermediates and disturbed the 13-cis-in equilibrium all-trans-isomerization.

An investigation of the electrochemical cycle of bacteriorhodopsin analogs with the modified ring.

5,6-Epoxy-, 4-methoxy-, 4-hydroxy-, and 3,4-dehydrobacteriorhodopsins can generate delta psi coupled to a photochemical cycle with intermediate M. The kinetics of delta psi comprises three main electrogenic phases: the fast small negative, the microsecond, and the millisecond positive phases. The photocycle efficiency is lower in all the analogs. The photocycle is modified insignificantly only in 3,4-dehydrobacteriorhodopsin. In the other pigments the decay of the flash-induced bleaching in the chromophore main absorption band is slower than the decay of M or long-wave intermediates, especially in the 4-hydroxy analog. In the latter analog, such distinctions, according to delta pH measurements, are partly due to deceleration of the decay of the novel intermediate (P). In 5,6-epoxybacteriorhodopsin, at all wavelengths, the decay of the intermediates takes seconds upon M formation. According to our and literature data, no bacteriorhodopsin analogs are known to have a cycle which preserves the M-intermediate and does not transport a proton.

[Synthesis and properties of C13-substituted retinals]. / Sintez i svoistva C13-zameshchennykh retinalei.

Several analogues of all-trans-retinal were synthesised, containing, instead of CH3-group at C13, the following substituents: H, C[2H]3, C2H5, iso-C3H7, C4H9, C6H5 or alpha-C10H8. The compounds synthesised on coupling with bacterioopsin gave artificial chromoproteins, which retained the ability to participate in the cycle of photochemical transformations and H+-transport.

The action of lanthanum ions and formaldehyde on the proton-pumping function of bacteriorhodopsin.

The photochemical cycle and the proton-pumping function of bacteriorhodopsin modified with lanthanum and formaldehyde has been studied. In both preparations, the M412 leads to BR570 transition time has been found to increase considerably. The deceleration of the photochemical cycle has been shown to be accompanied by inhibition of the millisecond phase of the photoelectrical response of bacteriorhodopsin membranes associated with phospholipid-impregnated collodion film. Photoelectrogenic activity measured with permeable ion probe in proteoliposomes was also inhibited. Effects of lanthanum were reversed by EDTA. Formation of M412 was slightly accelerated and the microsecond electrogenic phase was not affected by lanthanum and by formaldehyde. It is concluded that lanthanum, but not formaldehyde, can be used as a specific reversible inhibitor of the second half of the bacteriorhodopsin photocycle and of the associated H+ uptake on the cytoplasmic side of the halobacterial membrane. Possible mechanisms of these effects are discussed.

[Inhibition of bacteriorhodopsin by formalin and lanthanum]. / Ingibirovanie bakteriorodopsina formalinom i lantanom.

It was shown that the penetrating ions (PCB-) method can be used for quantitative estimation of the proton translocation function. Using this method it was found that the maximal inhibition (up to 5.5-6 times) can be achieved by 1% formalin treatment for 0.5 hrs in 0.5 M phosphate buffer, pH 7.8, 70 degrees. Using three methods, the kinetics of the obtained preparation were compared to those of the previously known inhibitor, La3+. The changes observed were as follows: inhibition of intermediate M412 decay in membrane suspension, decrease in the amplitude of the electric response millisecond phase in the purple membrane--collodium film system and deceleration of the overall photochemical cycle turnover in this system. The latter was registered by the rate of resporation of the electric response amplitude to the second light flash in the presence of uncoupling agents. The possible role of crosslinks during protein treatment with formalin is discussed.

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.

[Phases of photoelectrical response of bacteriorhodopsin]. / Fazy fotoélektricheskogo otveta bakteriorodopsina.

The generation of electrical potential difference by bacteriorhodopsin sheets incorporated into collodium film impregnated with a lecithine solution in decane was studied. The electrical response to a short light flash inducing a single turnover of bacteriorhodopsin consists of four phases. Phase I of photoresponse (tau less than 0.2 mks) has a small amplitude and is directed oppositely to the electrical response to continuous light. The negative phase is followed by positive phases, i.e. microsecond (II) and millisecond (III) ones, either of which can be decomposed at least into two exponents. Phase IV of the electrical response is a passive discharge of capacity of the "bacteriorhodopsin sheets--collodium film" system. Although phases II and III correspond to the generation and disappearance of the photochemical cycle intermediate with an absorption maximum at 412 nm, the parameters and activation energy of these two processes do not show complete coincidence. The effects of gramicidin A and the trifluoromethoxycarbonylcyanidephenylhydrazone uncoupler on the photoelectrical response were analyzed. The data obtained are discussed in terms of an equivalent electrical scheme, according to which the bacteriorhodopsin sheets form closed vesicles upon being incorporated into collodium film.

[Some features of photochemical conversions of bacteriorhodopsin at low pH values]. / Osobennosti fotokhimicheskikh prevrashchenii bakteriorodopsina pri nizkikh znacheniiakh pH.

Association of bacteriorhodopsin sheets and proteoliposomes with collodium film impregnated with a lecithine solution in decane was carried out. The generation of electrical potential difference in response to a light flash within the pH range of 0-6 was studied. The amplitude of microsecond (II) and millisecond (III) phases of the photoelectrical response was shown to decline with a decrease in pH as in the case when the inhibition by acid is due to protonation of a group with a pK of 3,7. The photoelectrical response of the "acid" form of bacteriorhodopsin with a maximun at 605 nm is represented by a negative phase having a small amplitude. This phase is coupled with the formation of the photochemical cycle bathointermediate and is sharply increased in an acid medium simultaneously with the generation of bacteriorhodopsin form with an absorption maximum at 565 nm. The effects of KCl, phenyldicarbaundecaborane and fluoride on the spectral and photoelectrical responses of bacteriorhodopsin are described.