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.

Electrogenic processes and protein conformational changes accompanying the bacteriorhodopsin photocycle.

The possible mechanisms of electrogenic processes accompanying proton transport in bacteriorhodopsin are discussed on the basis of recent structural data of the protein. Apparent inconsistencies between experimental data and their interpretation are considered. Special emphasis is placed on the protein conformational changes accompanying the reprotonation of chromophore and proton uptake stage in the bacteriorhodopsin photocycle.

Formation of the M(N) (M(open)) intermediate in the wild-type bacteriorhodopsin photocycle is accompanied by an absorption spectrum shift to shorter wavelength, like that in the mutant D96N bacteriorhodopsin photocycle.

Maximum of the M intermediate difference spectrum in the wild-type Halobacterium salinarium purple membrane is localized at 405-406 nm under conditions favoring accumulation of the M(N) intermediate (6 M guanidine chloride, pH 9.6), whereas immediately after laser flash the maximum is localized at 412 nm. The maximum is also localized at 412 nm 0.

Membrane potential stabilizes the O intermediate in liposomes containing bacteriorhodopsin.

In the bacteriorhodopsin-containing proteoliposomes, a laser flash is found to induce formation of a bathointermediate decaying in several seconds, the difference spectrum being similar to the purple-blue transition. Different pH buffers do not affect the intermediate, whereas an uncoupler, gramicidin A, and lipophilic ions accelerate decay of the intermediate or inhibit its formation. In the liposomes containing E204Q bacteriorhodopsin mutant, formation of the intermediate is suppressed.

Time-resolved generation of a membrane potential by ba3 cytochrome c oxidase from Thermus thermophilus. Evidence for reduction-induced opening of the binuclear center.

ba3-type cytochrome c oxidase purified from the thermophilic bacterium Thermus thermophilus has been reconstituted in phospholipid vesicles and laser flash-induced generation of a membrane potential by the enzyme has been studied in a microsecond/ms time scale with Ru(II)-tris-bipyridyl complex (RuBpy) as a photoreductant. Flash-induced single electron reduction of the aerobically oxidized ba3 by RuBpy results in two phases of membrane potential generation by the enzyme with tau values of about 20 and 300 microseconds at pH 8 and 23 degrees C. Spectrophotometric experiments show that oxidized ba3 reacts very poorly with hydrogen peroxide or any of the other exogenous heme iron ligands studied like cyanide, sulfide and azide.

Two forms of N intermediate (N(open) and N(closed)) in the bacteriorhodopsin photocycle.

Glutaraldehyde, aluminum ions and glycerol (that inhibit the M intermediate decay in the wild-type bacteriorhodopsin and azide-induced M decay in the D96N mutant by stabilization of the M(closed)) accelerate the N decay in the D96N mutant. The aluminum ions, the most potent activator of the N decay, induce a blue shift of the N difference spectrum by approximately 10 nm. Protonated azide as well as acetate and formate inhibit the N decay in both the D96N mutant and the wild-type protein.

Resolution of electrogenic steps coupled to conversion of cytochrome c oxidase from the peroxy to the ferryl-oxo state.

Charge translocation across the membrane coupled to transfer of the third electron in the reaction cycle of bovine cytochrome c oxidase (COX) has been studied. Flash-induced reduction of the peroxy intermediate (P) to the ferryl-oxo state (F) by tris-bipyridyl complex of Ru(II) in liposome-reconstituted COX is coupled to several phases of membrane potential generation that have been time-resolved with the use of an electrometric technique applied earlier in the studies of the ferryl-oxo-to-oxidized (F --> O) transition of the enzyme [Zaslavsky, D., et al.

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)).

Photovoltage evidence that Glu-204 is the intermediate proton donor rather than the terminal proton release group in bacteriorhodopsin.

Electrogenic events in the E204Q bacteriorhodopsin mutant have been studied. A two-fold decrease in the magnitude of microsecond photovoltage generation coupled to M intermediate formation in the E204Q mutant is shown. This means that deprotonation of E204 is an electrogenic process and its electrogenicity is comparable to that of the proton transfer from the Schiff base to D85.

Photophosphorylation in alkalophilic halobacterial cells containing halorhodopsin: chloride-ion cycle?

Light-driven ATP synthesis is found in cells of the alkalophilic bacterium Natronobacterium pharaonis containing halorhodopsin but deficient in H+-pumping bacteriorhodopsin. Photophosphorylation occurs with cyanide-inhibited respiratory chain as well as without cyanide in conditions with low C1- concentration in the incubation medium. Increase in C1- concentration from 0.

Flash-induced voltage changes in halorhodopsin from Natronobacterium pharaonis.

The flash-induced voltage response of halorhodopsin at high NaCl concentration comprises two main kinetic components. The first component with tau approximately 1 micros does not exceed 4% of the overall response amplitude and is probably associated with the formation of the L (hR520) intermediate. The second main component with tau approximately 1-2.

Cl- -dependent photovoltage responses of bacteriorhodopsin: comparison of the D85T and D85S mutants and wild-type acid purple form.

Laser flash-induced photovoltage responses of the D85S and D85T mutants as well as of the wild-type acid blue form are similar and reflect intraprotein charge redistribution caused by retinal isomerization. The Cl- -induced transition of all of these blue forms into purple ones is accompanied by the appearance of electrogenic stages, which is probably associated with Cl- translocation in the cytoplasmic direction. Cl- translocation efficiency of these purple forms is much lower than that of the proton transport by the wild-type bacteriorhodopsin.

The roles of the two proton input channels in cytochrome c oxidase from Rhodobacter sphaeroides probed by the effects of site-directed mutations on time-resolved electrogenic intraprotein proton transfer.

The crystal structures of cytochrome c oxidase from both bovine and Paracoccus denitrificans reveal two putative proton input channels that connect the heme-copper center, where dioxygen is reduced, to the internal aqueous phase. In this work we have examined the role of these two channels, looking at the effects of site-directed mutations of residues observed in each of the channels of the cytochrome c oxidase from Rhodobacter sphaeroides. A photoelectric technique was used to monitor the time-resolved electrogenic proton transfer steps associated with the photo-induced reduction of the ferryl-oxo form of heme a3 (Fe4+ = O2-) to the oxidized form (Fe3+OH-).

Inhibition of the M1-->M2 (M(closed) --> M(open)) transition in the D96N mutant photocycle and its relation to the corresponding transition in wild-type bacteriorhodopsin.

Glutaraldehyde, lutetium ions and glycerol inhibit the blue shift of the difference spectra maximum of the M intermediate in the D96N mutant. The M formed has a spectrum indistinguishable from the M intermediate in wild-type bacteriorhodopsin. It has been concluded that the M(open) form previously described by us is identical to the M2 and Mn intermediates postulated by Zimanyi et al.

Complicated character of the M decay pH dependence in the D96N mutant is due to the two pathways of the M conversion.

At high ionic strength, the pH dependence of the M intermediate decay in a photocycle of the D96N mutant bacteriorhodopsin shows a complicated behavior which is found to be due to the coexistence of two pathways of the M conversion. The M decay which dominates at pH < 5 is coupled to the proton uptake from the cytoplasmic surface and proceeds probably through the N intermediate. This pathway is inhibited by glutaraldehyde, the potent inhibitor of M decay in the wild-type bacteriorhodopsin and of the azide-facilitated M decay in the D96N mutant.

Two bacteriorhodopsin M intermediates differing in accesibility of the Schiff base for azide.

Glutaraldehyde treatment leads to the inhibition (i) of the M intermediate decay in wild-type bacteriorhodopsin (bR) and (ii) of the azide-facilitated M decay in the D96N mutant bR. LuCl3 is shown to be a more potent inhibitor of both processes. Glycerol and sucrose are also inhibitors.

delta psi-mediated signalling in the bacteriorhodopsin-dependent photoresponse.

It has been shown previously that the proton-pumping activity of bacteriorhodopsin from Halobacterium salinarium can transmit an attractant signal to the bacterial flagella upon an increase in light intensity over a wide range of wavelengths. Here, we studied the effect of blue light on phototactic responses by the mutant strain Pho8l-B4, which lacks both sensory rhodopsins but has the ability to synthesize bacteriorhodopsin. Under conditions in which bacteriorhodopsin was largely accumulated as the M412 bacteriorhodopsin photocycle intermediate, halobacterial cells responded to blue light as a repellent.

Cooperative phenomena in the photocycle of D96N mutant bacteriorhodopsin.

The M intermediate decay in the photocycle of D96N mutant bacteriorhodopsin does not depend on the light intensity of the exciting flash. Cooperative phenomena in the photocycle are revealed after addition of azide causing acceleration of the M decay and making it kinetically well separated from the N decay. Increase in the light intensity induces slight deceleration of the M decay and significant acceleration of the N decay.

M-decay in the bacteriorhodopsin photocycle: effect of cooperativity and pH.

The dependence of the bacteriorhodopsin (bR) photocycle on the intensity of the exciting flash was investigated in purple membranes. The dependence was most pronounced at slightly alkaline pH values. A comparison study of the kinetics of the photocycle and proton uptake at different intensities of the flash suggested that there exist two parallel photocycles in purple membranes at a high intensity of the flash.

Rapid kinetics of membrane potential generation by cytochrome c oxidase with the photoactive Ru(II)-tris-bipyridyl derivative of cytochrome c as electron donor.

Yeast iso-1-cytochrome c covalently modified at cysteine-102 with (4-bromomethyl-4'-methylbipyridine)[bis(bipyridine)]Ru2+ (Ru-102-Cyt c) has been used as a photoactive electron donor to mitochondrial cytochrome c oxidase (COX) reconstituted into phospholipid vesicles. Rapid kinetics of membrane potential generation by the enzyme following flash-induced photoreduction of Ru-102-Cyt c heme has been measured and compared to photovoltaic responses observed with Ru(II)(bipyridyl)3 (RuBpy) as the photoreductant [D.L.

On the two forms of bacteriorhodopsin.

In our previous work [(1993) FEBS Lett. 313, 248-250; (1993) Biochem. Int.

Ion permeability induced in artificial membranes by the ATP/ADP antiporter.

The hypothesis on the additional function of the ATP/ADP antiporter (ANT) as uncoupling protein has been tested in proteoliposomes and planar bilayer phospholipid membranes (BLM). It is found that dissipation of the light-induced delta pH in the dark is very much faster in ANT-bacteriorhodopsin proteoliposomes than in proteoliposomes containing bacteriorhodopsin as the only protein. Mersalyl treatment of ANT-bacteriorhodopsin proteoliposomes causes further increase in the delta pH dissipation rate due to formation of a high conductance pore.

Flash-induced membrane potential generation by cytochrome c oxidase.

Flash-induced single-electron reduction of cytochrome c oxidase. Compound F (oxoferryl state) by RuII(2,2'-bipyridyl)3(2+) [Nilsson (1992) Proc. Natl.

Bacteriorhodopsin is involved in halobacterial photoreception.

The bacterio-opsin gene was introduced into a "blind" Halobacterium salinarium mutant that (i) lacked all the four retinal proteins [bacteriorhodopsin (BR), halorhodopsin, and sensory rhodopsins (SRs) I and II] and the transducer protein for SRI and (ii) showed neither attractant response to long wavelength light nor repellent response to short wavelength light. The resulting transformed cells acquired the capability to sense light stimuli. The cells accumulated in a light spot, demonstrating the BR-mediated orientation in spatial light gradients.

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.