On the kinetics of voltage formation in purple membranes of Halobacterium salinarium.

The kinetics of the bacteriorhodopsin photocycle, measured by voltage changes in a closed membrane system using the direct electrometrical method (DEM) of Drachev, L.A., Jasaitus, A.A., Kaulen, A.D., Kondrashin, A.A., Liberman, E.A., Nemecek, I.B., Ostroumov, S.A., Semenov, Yu, A. & Skulachev, V.P. (1974) Nature 249, 321-324 are sixfold slower than the kinetics obtained in optical studies with suspensions of purple membrane patches. In this study, we have investigated the reasons for this discrepancy. In the presence of the uncouplers carbonyl cyanide m-chlorophenylhydrazone or valinomycin, the rates in the DEM system are similar to the rates in suspensions of purple membrane. Two alternative explanations for the effects of uncouplers were evaluated: (a) the 'back-pressure' of the Deltamicro;H+ slows the kinetic steps leading to its formation, and (b) the apparent difference between the two systems is due to slow major electrogenic events that produce little or no change in optical absorbance. In the latter case, the uncouplers would decrease the RC time constant for membrane capacitance leading to a quicker discharge of voltage and concomitant decrease in photocycle turnover time. The experimental results show that the primary cause for the slower kinetics of voltage changes in the DEM system is thermodynamic back-pressure as described by Westerhoff, H.V. & Dancshazy, Z. (1984) Trends Biochem. Sci. 9, 112-117.

Reduction and protonation of the secondary quinone acceptor of Rhodobacter sphaeroides photosynthetic reaction center: kinetic model based on a comparison of wild-type chromatophores with mutants carrying Arg-->Ile substitution at sites 207 and 217 in the L-subunit.

After the light-induced charge separation in the photosynthetic reaction center (RC) of Rhodobacter sphaeroides, the electron reaches, via the tightly bound ubiquinone QA, the loosely bound ubiquinone Q(B) After two subsequent flashes of light, Q(B) is reduced to ubiquinol Q(B)H2, with a semiquinone anion Q-(B) formed as an intermediate after the first flash. We studied Q(B)H2 formation in chromatophores from Rb. sphaeroides mutants that carried Arg-->Ile substitution at sites 207 and 217 in the L-subunit. While Arg-L207 is 17 A away from Q(B), Arg-L217 is closer (9 A) and contacts the Q(B)-binding pocket. From the pH dependence of the charge recombination in the RC after the first flash, we estimated deltaG(AB), the free energy difference between the Q-(A)Q(B) and Q(A)Q-(B) states, and pK212, the apparent pK of Glu-L212, a residue that is only 4 A away from Q(B). As expected, the replacement of positively charged arginines by neutral isoleucines destabilized the Q-(B) state in the L217RI mutant to a larger extent than in the L207RI one. Also as expected, pK212 increased by approximately 0.4 pH units in the L207RI mutant. The value of pK212 in the L217RI mutant decreased by 0.3 pH units, contrary to expectations. The rate of the Q-(A)Q-(B)-->Q(A)Q(B)H2 transition upon the second flash, as monitored by electrometry via the accompanying changes in the membrane potential, was two times faster in the L207RI mutant than in the wild-type, but remained essentially unchanged in the L217RI mutant. To rationalize these findings, we developed and analyzed a kinetic model of the Q-(A)Q-(B)-->Q(A)Q(B)H2 transition. The model properly described the available experimental data and provided a set of quantitative kinetic and thermodynamic parameters of the Q(B) turnover. The non-electrostatic, 'chemical' affinity of the QB site to protons proved to be as important for the attracting protons from the bulk, as the appropriate electrostatic potential. The mutation-caused changes in the chemical proton affinity could be estimated from the difference between the experimentally established pK2J2 shifts and the expected changes in the electrostatic potential at Glu-L212, calculable from the X-ray structure of the RC. Based on functional studies, structural data and kinetic modeling, we suggest a mechanistic scheme of the QB turnover. The detachment of the formed ubiquinol from its proximal position next to Glu-L212 is considered as the rate-limiting step of the reaction cycle.

Electrogenicity at the donor/acceptor sides of cyanobacterial photosystem I.

To study electrogenesis the photosystem I particles from Synechococcus elongatus were incorporated into asolectin liposomes, and fast kinetics of laser flash-induced electric potential difference generation has been measured by a direct electrometric method in proteoliposomes absorbed on a phospholipid-impregnated collodion film. The photoelectric response has been found to involve three electrogenic stages associated with (i) iron-sulfur center Fx reduction by the primary electron donor P700, (ii) electron transfer between iron-sulfur centers Fx and FA/FB, and (iii) reduction of photo-oxidized P700+ by reduced cytochrome C553. The relative magnitudes of phases (ii) and (iii) comprised about 20% of phase (i).

Two o-type oxidases in Methylobacillus flagellatum KT.

Two oxidases of the o-type in membranes of the methanol-grown obligate methylotroph Methylobacillus flagellatum KT were distinguished. For this purpose the kinetic analysis of the laser flash-induced optical absorbance changes of CO-oxidase complexes under reducing conditions was used. The ratio of these oxidases in membranes greatly depended on the phases of bacterial growth. One of the oxidases appeared to belong to the Escherichia coli o-type oxidase family being more sensitive to KCN (Ki = 1 microM). It showed monophasic CO recombination kinetics with tau 25-30 ms and was expressed in the early exponential phase of growth. The other oxidase seemed to be similar to the Bacillus sp. FTU o-type oxidase being less sensitive to KCN (Ki = 6 microM), having three-phasic CO reassociation kinetics with tau 35-70 microseconds, 0.25-0.5 ms and 2-4 ms and dominating in the stationary growth phase. Pyridine haemochrome spectra showed haems A and D to be absent from the bacterial membranes.

pH dependence of the formation of an M-type intermediate in the photocycle of 13-cis-bacteriorhodopsin.

An M-type intermediate is formed in the 13-cis-bR photocycle in purple membranes at high pH. This is presumably due to deprotonation of the same group whose deprotonation causes a large increase in rate of M formation in the trans-bR photocycle (the 'alkaline transition'). For Triton X-100-solubilized bR, the alkaline transition is shifted to a lower pH value by more than 2 pH units. The alkaline transition in Triton-solubilized preparations changes the efficiency of the M intermediate formation in the 13-cis-sbR photocycle. The M intermediate formation in 13-cis-sbR, as in the case of trans-sbR, is completely inhibited when the blue 'acidic' bR is formed at low pH. The protonation state of the group affecting formation of the M intermediate in 13-cis-bR at high pH and the group which is responsible for the transition to the blue acidic form influence in a similar way the equilibrium between bR isomers in the dark-adapted form as well as the rate of dark adaptation.

Kinetics of CO binding to putative Na(+)-motive oxidases of the o-type from Bacillus FTU and of the d-type from Escherichia coli.

The kinetics of CO reassociation with isolated Bacillus FTU o-type oxidase and with solubilized membranes of Escherichia coli (GO102 strain) containing the d-type oxidase only, upon laser flash photolysis under reducing conditions, were studied. In both cases, kinetics are shown to be composed of three phases (tau 35-70 microseconds, 0.25-0.5 ms and 2-5 ms). The spectra of the flash-induced absorbance changes of the first kinetic components proved to be characteristic of CO-o- and CO-b595 d-cytochrome complexes in Bac. FTU and E. coli, respectively. The spectra of the second and the third components appeared to be nearly the same in Bac. FTU and E. coli with peaks for the former at 436-437 and 590 nm and troughs at 419-420 and 569 nm; and for the latter with peaks at 436-437 and 558-560 nm and troughs at 419-420 and 575-578 nm. The similarity between the putative Na(+)-pumping Bac. FTU o- and E. coli d-type oxidases and their difference from the H(+)-motive Bac. FTU caa3- and E. coli o-type oxidases are discussed.

Kinetics of CO binding to H(+)-motive oxidases of the caa3-type from Bacillus FTU and of the o-type from Escherichia coli.

The kinetics of CO rebinding with isolated Bacillus FTU caa3-type oxidase and with solubilized Escherichia coli membranes (GO103 strain) containing the o-type oxidase as the main O2-reducing enzyme were studied under reducing conditions by laser flash photolysis of the CO-oxidase complexes. The spectra of the optical absorbance changes upon photolysis were characteristic of CO-caa3- and CO-o-oxidase complexes in Bac. FTU and E. coli, respectively. Small quantities of d-type oxidase in E. coli GO103 membranes were detected. The kinetics of CO reassociation with reduced caa3- and o-type oxidases were monophasic with tau 25-30 ms in both cases.

On the two pathways of the M-intermediate formation in the photocycle of bacteriorhodopsin.

The flash-photolysis technique was used to study the photocycles of the wild-type bacteriorhodopsin (WT bR) and D96N mutant. Kinetics of the L-intermediate decay and M-intermediate formation at pH 7.0, 20 degrees C fit well a sum of two components having time constants, tau (1) = 60 microS and tau (2) = 250 microS, for the WT bR, and a sum of three components having time constants, tau (1) = 55 microS, tau (2) = 220 microS and tau (3) = 1 mS, for the D96N mutant. The fast component with a time constant of 1.4 microS was found in the photoresponse at 400 nm. It constituted 10% of the total amplitude and may be attributed to the K-->L transition. The component with tau = 1 mS was observed in the photocycle of the WT bR as a lag phase in the relaxation of the photoresponse at 400 nm. The difference absorbance minima, corresponding to the first (55-60 microS) and the second (220-260 microS) components of the M-formation, were located at 550 and 530 nm, respectively. The absorbance spectra, corresponding to the 1-mS-component of the M-formation of the D96N bR, may be represented as a superposition of spectra, corresponding to the first and the second components in the region of 460-700 nm. The effect of azide on the D96N bR revealed two azide-independent components in the decay of L-intermediate. Azide was shown to protonate all M-forms simultaneously. This indicates that the Schiff base pK rises almost immediately after deprotonation.(ABSTRACT TRUNCATED AT 250 WORDS)

Interrelations of M-intermediates in bacteriorhodopsin photocycle.

The photocycles of the wild-type bacteriorhodopsin and the D96N mutant were investigated by the flash-photolysis technique. The M-intermediate formation (400 nm) and the L-intermediate decay (520 nm) were found to be well described by a sum of two exponents (time constants, tau 1 = 65 and tau 2 = 250 microseconds) for the wild-type bR and three exponents (tau 1 = 55 microseconds, tau 2 = 220 microseconds and tau 3 = 1 ms) for the D96N mutant of bR. A component with tau = 1 ms was found to be present in the photocycle of the wild-type bacteriorhodopsin as a lag-phase in the relaxation of photoresponses at 400 and 520 nm. In the presence of Lu3+ ions or 80% glycerol this component was clearly seen as an additional phase of M-formation. The azide effect on the D96N mutant of bR suggests that the 1-ms component is associated with an irreversible conformational change switching the Schiff base from the outward to the inward proton channel. The maximum of the difference spectrum of the 1-ms component of D96N bR is located at 404 nm as compared to 412 nm for the first two components. We suggest that this effect is a result of the alteration of the inward proton channel due to the Asp96-->Asn substitution. Proton release measured with pyranine in the absence of pH buffers was identical for the wild-type bR and D96N mutant and matched the M-->M' conformational transition. A model for M rise in the bR photocycle is proposed.

[Study of intermediate N using mutant forms of bacteriorhodopsin at Asp-96]. / Issledovanie intermediatea N s pomoshch'iu mutantnykh form bakteriorhodopsina po Asp-96.

It has been found that the N(P, R)-type intermediate of the photocycle is formed in the Asp-96-->Asn mutant at acidic pH. Azide, which strongly activates the M decay in this mutant, allows the N intermediate to be shown also at neutral pH. Under these conditions mutant N decays in a pH-independent fashion. In the presence of azide, the H+ uptake by Asp-96-->Asn mutant bacteriorhodopsin follows the M decay, whereas the N decay occurs at a much slower rate. Two electrogenic stages have been shown to be associated with the M--->bR step in the Asp-96--->Asn mutant photocycle. The faster and slower stages correlate with the M--->N and N--->bR transitions, respectively. In the Asp-96--->Asn mutant, high concentrations of azide are found to increase the M decay rate up to the values higher than those in the wild-type protein, both with or without azide. Such an effect is absent for the Asp-96-->Glu mutant. The activation energies for M--->N and N--->bR transitions in the wild-type protein are equal to 18 and 19 kcal x mole-1, respectively. In the Asp-96-->Asn mutant without azide, the activation energy of the M decay is only 5 kcal x mole-1, whereas in the presence of azide in this mutant the activation energies for M and N decays are 8 and 9 kcal x mole-1, respectively. A scheme of events accompanying the Schiff base reprotonation during the photocycle is discussed.

Functioning of quinone acceptors in the reaction center of the green photosynthetic bacterium Chloroflexus aurantiacus.

The photosynthetic reaction centers (RC) of the green bacterium Chloroflexus aurantiacus have been investigated by spectral and electrometrical methods. In these reaction centers, the secondary quinone was found to be reconstituted by the addition of ubiquinone-10. The equilibrium constant of electron transfer between primary (QA) and secondary (QB) quinones was much higher than that in RC of purple bacteria. The QB binding to the protein decreased under alkalinization with apparent pK 8.8. The single flash-induced electric responses were about 200 mV. An additional electrogenic phase due to the QB protonation was observed after the second flash in the presence of exogenous electron donors. The magnitude of this phase was 18% of that related to the primary dipole (P+QA-) formation. Since the C. aurantiacus RC lacks H-subunit, this subunit was not an obligatory component for electrogenic QB protonation.

Slow electrogenic events in the cytochrome bc1-complex of Rhodobacter sphaeroides. The electron transfer between cytochrome b hemes can be non-electrogenic.

The flash-induced formation of transmembrane electric potential differences (measured by carotenoid bandshift) and redox changes of cytochrome bh (b561) were monitored spectrophotometrically in Rb. sphaeroides chromatophores in a pH range from 7.5 to 10.0. It is shown that in the presence of antimycin A and at pH less than 8.3 the myxothiazol-sensitive, antimycin-insensitive component of the carotenoid bandshift is kinetically coupled to cytochrome bh reduction. The kinetics of both processes can be described by a single exponent with a rise time of about 10 ms. Alkalization of the medium (8.3 less than or equal to pH less than or equal to 9.2) causes the appearance of an additional constituent in this phase of the carotenoid response with the rise time varying in the range of 100-300 ms. With a further pH increase (pH greater than 9.2), the electrogenic constituent, kinetically linked to cytochrome bh reduction, diminishes. The obtained data are discussed within the framework of the scheme, assuming that the electron transfer between bl and bh hemes in the bc1 complex is, under certain conditions, accompanied by proton transfer in the same direction.

Partial reversion of the electrogenic reaction in the ubiquinol: cytochrome c2-oxidoreductase of Rhodobacter sphaeroides chromatophores under neutral and alkaline conditions.

The interaction of the photosynthetic reaction center (RC)-generated ubiquinol with the ubiquinone-reducing center C of ubiquinol:cytochrome c2-oxidoreductase (bc1-complex) has been studied electrometrically in Rhodobacter sphaeroides chromatophores. The addition of myxothiazol inhibited the ubiquinol-oxidizing center Z, suppressing the phases of membrane potential generation by the bc1-complex, but at the same time induced an electrogenic phase of opposite polarity, sensitive to antimycin A, the inhibitor of center C. The rise time of this reverse phase varied from 3 ms at pH 6.0 to 1 ms at pH 9.5. At pH greater than 9.5 the reverse phase was limited by the rate of ubiquinol formation in RC. The magnitude of the reverse phase was constant within the pH range 7.5-10.0. It is assumed that the reverse phase is due to the electrogenic deprotonation reaction which takes place after the binding of the RC-generated ubiquinol to center C.

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.

Orientation of reaction center complexes from Rhodobacter sphaeroides in proteoliposomes and the effect of o-phenanthroline on electrogenesis during primary photochemical reaction.

The orientation of Rhodobacter sphaeroides reaction center complexes (RC complexes) in proteoliposomal membranes was investigated by a direct electrometric method. Conditions were found that allow monitoring of only that RC complex fraction that is oriented with its donor side to the inner part of the proteoliposome. It is shown that o-phenanthroline, an inhibitor of electron transfer between primary (QA) and secondary (QB) quinone acceptors, can also inhibit the photoinduced QA reduction. The efficiency of this inhibition depends on the concentration of added ubiquinone. It is assumed that the laser flash-induced o-phenanthroline inhibition of primary dipole (P-870+.QA-) formation is of a competitive nature.

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.

Replacement of aspartic acid-96 by asparagine in bacteriorhodopsin slows both the decay of the M intermediate and the associated proton movement.

The photocycle, electrical charge translocation, and release and uptake of protons from the aqueous phase 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 neutral pH the main effect of the Asp-96----Asn mutation is to slow by 2 orders of magnitude the decay of the M intermediate and the concomitant charge displacement associated with the reprotonation of the Schiff base from the cytoplasmic side of the membrane. The proton uptake measured with the indicator dye pyranine is likewise slowed without affecting the stoichiometry of proton pumping. The corresponding results for the Asp-96----Glu mutant, on the other hand, are very close to those for the wild-type protein. These results provide a kinetic explanation for the fact that at pH 7 and saturating light intensities the steady-state proton pumping is almost abolished in the Asp-96----Asn mutant but is close to normal in the Asp-96----Glu mutant. Thus, the pump is simply turning over much more slowly in the Asp-96----Asn mutant. The time constants of the decay of M and the associated charge translocation increase strongly with increasing pH for the Asp-96----Asn mutant but are virtually pH-independent for the Asp-96----Glu mutant and wild-type bacteriorhodopsin. At pH 5 the M decay of the Asp-96----Asn mutant is as fast as for wild type. These results suggest that Asp-96 serves as an internal proton donor in the proton-uptake pathway from the cytoplasm to the Schiff base.

Electrogenic steps in the redox reactions catalyzed by photosynthetic reaction-centre complex from Rhodopseudomonas viridis.

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 alpha absorption maxima at 554 nm, 552 nm and 556 nm respectively. The 380-mV heme displays a split alpha band with a maximum at 559 nm and a shoulder at 552 nm. Such a splitting is due to non-degenerated Qx and Qy transitions in the iron-porphyrin 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+ (tau = 0.32 microsecond) whereas heme c-556 serves to rereduce c-559 (tau = 2.5 microsecond). Upon reduction of the third heme (c-552), the P-960+ reduction rate increases twofold (tau = 0.17 microsecond) and all photoinduced redox events within the cytochrome appear to be complete in less than 1 microsecond 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-ns 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; (II) reduction of P-960+ by heme c-559; (III) reduction of c-559 by c-556 and (IV) protonation of Q2-B. The relative contributions of stages I, II, III and IV are found to be equal to 70%, 15%, 5% and 10%, 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.(ABSTRACT TRUNCATED AT 400 WORDS)

Proteinase-treated photoreceptor discs. Photoelectric activity of the partially-digested rhodopsin and membrane orientation.

Photoreceptor discs from rod outer segments of cattle retina were treated with (a) papain, (b) thermolysin or (c) trypsin, the procedures resulting in the cleavage of the rhodopsin polypeptide chain between (a) 323 and 324, 236 and 237, 241 and 242, (b) 327 and 328, 240 and 241, or (c) 339 and 340 amino acid residues, respectively. In all the cases, partially digested rhodopsins proved to be competent in generating photoelectric potential and increasing membrane conductance of the discs adsorbed onto phospholipid-impregnated collodion film. The kinetics of generation and dissipation of photopotential as well as of formation of metarhodopsin II and of the light-induced rhodopsin protonation were found to be the same in the partially digested preparations and in the intact one. Incubation of papain-treated or thermolysin-treated discs at pH 6.0 induced formation of inside-out vesicles which, when incorporated into the collodion film, generated an oppositely directed photopotential. Treatment of such vesicles with papain gave rise to further cleavages of the polypeptide localized between 30 and 31, 186 and 187 amino acid residues. One more proteinase-sensitive site, localized between 104 and 105 residues, has been discovered in the inside-out vesicles treated with thermolysin. This fact consistent with the scheme of the 'seven column' arrangement of the visual rhodopsin [Ovchinnikov, Yu. A. (1982) FEBS Lett. 148, 179-191]. Rhodopsin, when treated with papain on both sides, was deprived of sixty amino acid residues being split in two sites in the middle part of the polypeptide, but was still active as a photoelectric energy transducer. The main specific feature inherent in the photoelectric response of the papain-treated or thermolysin-treated rhodopsin and absent from the native protein is that the former survives addition of long trains of saturating flashes when the response of the intact preparation becomes negligible. This effect was shown to be due to conversion of partially digested rhodopsin to a photolytic product that at room temperature lived for minutes even in the presence of NH2OH. A 532-nm laser flash effectively converted this product back to rhodopsin.