Distortion of the L-->M transition in the photocycle of the bacteriorhodopsin mutant D96N: a time-resolved step-scan FTIR investigation

The D96N mutant of bacteriorhodopsin has often been taken as a model system to study the M intermediate of the wild type photocycle due to the long life time of the corresponding intermediate of the mutant. Using time-resolved step-scan FTIR spectroscopy in combination with a sample changing wheel we investigated the photocycle of the mutant with microsecond time resolution. Already after several microseconds an intermediate similar to the MN state is observed, which contrasts with the M state of the wild type protein. At reduced hydration M and N intermediates similar to those of wild type BR can be detected. These results have a bearing on the interpretation of the photocycle of this mutant. A mechanism is suggested for the fast rise of MN which provides some insight into the molecular events involved in triggering the opening of the cytosolic channel also of the wild type protein.     

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 0.1 msec after the flash in the absence of guanidine chloride at pH 11.3. Within several milliseconds the maximum is shifted to short-wavelength region by 5-6 nm. This shift is similar to that in the D96N mutant which accompanies the M(N) (M(open)) intermediate formation. The main two differences are: 1) the rate of the shift is slower in the wild-type bacteriorhodopsin, and is similar to the rate of the M to N intermediate transition (t1/2 approximately 2 msec); 2) the shift in the wild-type bacteriorhodopsin is observed at alkaline pH values which are higher than pK of the Schiff base (approximately 10.8 at 1 M NaCl) in the N intermediate with the deprotonated Asp-96. Thus, the M(N) (M(open)) intermediate with open water-permeable inward proton channel is observed only at high pH, when the Schiff base and Asp-96 are deprotonated. The data confirmed our earlier conclusion that the M intermediate observed at lower pH has the closed inward proton channel.     

Study of intermediate N using mutant forms of bacteriorhodopsin at 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.     

Deriving the intermediate spectra and photocycle kinetics from time-resolved difference spectra of bacteriorhodopsin. The simpler case of the recombinant D96N protein.

The bacteriorhodopsin photocycle contains more than five spectrally distinct intermediates, and the complexity of their interconversions has precluded a rigorous solution of the kinetics. A representation of the photocycle of mutated D96N bacteriorhodopsin near neutral pH was given earlier (Váró, G., and J. K. Lanyi. 1991. Biochemistry. 30:5008-5015) as BRhv-->K<==>L<==>M1-->M2--> BR. Here we have reduced a set of time-resolved difference spectra for this simpler system to three base spectra, each assumed to consist of an unknown mixture of the pure K, L, and M difference spectra represented by a 3 x 3 matrix of concentration values between 0 and 1. After generating all allowed sets of spectra for K, L, and M (i.e., M1 + M2) at a 1:50 resolution of the matrix elements, invalid solutions were eliminated progressively in a search based on what is expected, empirically and from the theory of polyene excited states, for rhodopsin spectra. Significantly, the average matrix values changed little after the first and simplest of the search criteria that disallowed negative absorptions and more than one maximum for the M intermediate. We conclude from the statistics that during the search the solutions strongly converged into a narrow region of the multidimensional space of the concentration matrix. The data at three temperatures between 5 and 25 degrees C yielded a single set of spectra for K, L, and M; their fits are consistent with the earlier derived photocycle model for the D96N protein.     

Temperature jump study of charge translocation during the bacteriorhodopsin photocycle

Temperature jump experiments were carried out on purple membranes oriented and fixed in polyacrylamide gel. With green background illumination a relaxation of the photocurrent after an infrared laser pulse could be observed. To simulate the temperature jump signals different models of the bacteriorhodopsin photocycle were tested. The parameters of these models were obtained by measuring absorbance changes and photocurrent after excitation with a 575-nm laser flash.

A model with a temperature-dependent branching before the M state turned out to be satisfying. Other models, especially those with a late branching or without branching, could not reproduce the temperature jump measurements.

     

Photoreaction of N560 intermediate in the photocycle of bacteriorhodopsin.

Sophisticated measurements were made on the nanosecond time-resolved absorbance change of the purple membrane of Halobacterium halobium under cw background light irradiation (440-800 nm, 11-441 mW/cm2). A red-shifted transient species R660 (KN, Q) was found in alkaline conditions (pH > 9.3). Background light intensity effect shows that (i) R660 is photochemically formed from N560 intermediate which is accumulated under background light irradiation because of the elongated lifetime in alkaline suspension, and that (ii) the slow decaying M412 is not photochemically formed from N560 but from bR568.     

Crystal structure of the D85S mutant of bacteriorhodopsin: model of an O-like photocycle intermediate.

Crystal structures are reported for the D85S and D85S/F219L mutants of the light-driven proton/hydroxyl-pump bacteriorhodopsin. These mutants crystallize in the orthorhombic C222(1) spacegroup, and provide the first demonstration that monoolein-based cubic lipid phase crystallization can support the growth of well-diffracting crystals in non-hexagonal spacegroups. Both structures exhibit similar and substantial differences relative to wild-type bacteriorhodopsin, suggesting that they represent inherent features resulting from neutralization of the Schiff base counterion Asp85. We argue that these structures provide a model for the last photocycle intermediate (O) of bacteriorhodopsin, in which Asp85 is protonated, the proton release group is deprotonated, and the retinal has reisomerized to all-trans. Unlike for the M and N photointermediates, where structural changes occur mainly on the cytoplasmic side, here the large-scale changes are confined to the extracellular side. As in the M intermediate, the side-chain of Arg82 is in a downward configuration, and in addition, a pi-cloud hydrogen bond forms between Trp189 NE1 and Trp138. On the cytoplasmic side, there is increased hydration near the surface, suggesting how Asp96 might communicate with the bulk during the rise of the O intermediate.     

Primary charge motions and light-energy transduction in bacteriorhodopsin

The bacteriorhodopsin protein (bR) in the cell membrane of Halobacterium halobium is a light driven proton pump. Many details are known about its structure and the molecular mechanism of proton translocation. The events may be characterized by: (1) the changes in light absorption after photon excitation (the photocycle); (2) the charge motion cycle inside the protein: the steps taken by the proton during translocation; (3) the retinal cycle. changes in isomerization and protonation; and (4) the opsin cycle: alterations of protonation of different amino acids in the apoprotein. From a review of existing data a more or less concise picture of the parallelism of the above four cycles emerges, which may be valuable as a model for understanding other types of molecular pumps.     

Late events in the photocycle of bacteriorhodopsin mutant L93A

In the photocycle of bacteriorhodopsin (bR) from Halobacterium salinarum mutant L93A, the O-intermediate accumulates and the cycling time is increased approximately 200 times. Nevertheless, under continuous illumination, the protein pumps protons at near wild-type rates. We excited the mutant L93A in purple membrane with single or triple laser flashes and quasicontinuous illumination, (i.e., light for a few seconds) and recorded proton release and uptake, electric signals, and absorbance changes. We found long-living, correlated, kinetic components in all three measurements, which-with exception of the absorbance changes-had not been seen in earlier investigations. At room temperature, the O-intermediate decays to bR in two transitions with rate constants of 350 and 1800 ms. Proton uptake from the cytoplasmic surface continues with similar kinetics until the bR state is reestablished. An analysis of the data from quasicontinuous illumination and multiple flash excitation led to the conclusion that acceleration of the photocycle in continuous light is due to excitation of the N-component in the fast N<-->O equilibrium, which is established at the beginning of the severe cycle slowdown. This conclusion was confirmed by an action spectrum.     

Thermal equilibration between the M and N intermediates in the photocycle of bacteriorhodopsin.

The stages in the photocycle of bacteriorhodopsin (BR) involving the M and N intermediates are investigated using a double pulse excitation method. A first (cycling) pulse at 532 nm is followed, with an appropriate time delay, by a second pulse (337, 406, 446, or 470 nm) which induces the M-->BR back-photoreaction. After depletion by the second pulse a repopulation of M in the millisecond range is observed which is interpreted in terms of a thermal N-->M relaxation. It is thus concluded that a (thermal) M<-->N equilibrium accounts for the biphasic decay of M in the BR photocycle. Other models for this stage of the light-driven proton-pump are therefore unnecessary.     

The quantum efficiency of the bacteriorhodopsin photocycle.

The quantum yield of the primary photoprocess in light-adapted bacteriorhodopsin (phi 1) was determined at room temperature with low-intensity 530 nm neodymium laser excitation, with bovine rhodopsin as a relative actinometer. The observed value of phi 1 - 0.25 +/- 0.05, and the previously determined parameter phi 1/phi 2 - 0.4 [where phi 2 denotes the quantum efficiency of the back photoprecess from the primary species K (590)] imply that phi 1 + phi 2 approximately equal 1. This feature, also characterizing the photochemistry of rhodopsin, bears on the nature and mechanism of the primary event in both systems.     

The residues Leu 93 and Asp 96 act independently in the bacteriorhodopsin photocycle: studies with the leu 93-->Ala, Asp 96-->Asn double mutant.

Previous mutagenesis studies with bacteriorhodopsin have shown that reprotonation of the Schiff's base is the rate-limiting step in the photocycle of the D96N mutant, whereas retinal re-isomerization and return of the protein to the initial state constitute the rate-limiting events in the photocycle of the L93A mutant. Thus, in the D96N mutant, decay of the M intermediate is slowed down by more than 100-fold at pH 7. In the L93A mutant, decay of the O intermediate is slowed down by 250-fold. We report here that in the L93A, D96N double mutant, decay of the M intermediate, as well as the formation and decay of the O intermediate, are slowed down dramatically. The photocycle is completed by the decay of a long-lived O intermediate, as in the L93A mutant. The decay of the M and O intermediates in the double mutant parallels the behavior seen in the single mutants over a wide temperature and pH range, arguing that the observed independence is an intrinsic property of the mutant. The slow decay of the M and O intermediates can be selectively and independently reversed under conditions identical to those used for the corresponding intermediates in the D96N and L93A single mutants. Because the effects of the two individual mutations are preserved in the double mutant and can be independently reversed, we conclude that residues Asp 96 and Leu 93 act independently and at different stages of the bacteriorhodopsin photocycle. These results also show that formation of the O intermediate only requires protonation of the Schiff's base and is independent of the protonation of Asp 96 from the aqueous medium.     

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. It is concluded that the N intermediate represents, in fact, an equilibrium mixture of the two ('open' and 'closed') forms. These two forms, like M(closed) and M(open), come to an equilibrium in the microseconds range. The absorption spectrum of the N(open) is slightly shifted to red in comparison to that of the N(closed). Again, this resembles the M forms. 13-cis-all-trans re-isomerization is assumed to occur in the N(closed) form only. Binding of 1-2 molecules of protonated azide stabilizes the N(open) form. Existence of the 'open' and 'closed' forms of the M and N intermediates provides the appropriate explanation of the cooperative phenomenon as well as some other effects on the bacteriorhodopsin photocycle. Summarizing the available data, we suggest that M(open) is identical to the M(N) form, whereas M1 and M2 are different substates of M(closed).     

Two processes lead to a stable all-trans and 13-cis isomer equilibrium in dark-adapted bacteriorhodopsin; effect of high pressure on bacteriorhodopsin,bacteriorhodopsin mutant D96N and fluoro-bacteriorhodopsin analogues

The combination of absorption spectroscopy and extraction techniques was applied to study the effect of high pressure on the dark-adapted state of bacteriorhodopsin, 14-(12-,10-)fluoro-bacteriorhodopsin, a D96N bacteriorhodopsin mutant, and 14-(12-,10-)fluoro-D96N. Evidence is presented that, at high pressure, the isomers' equilibrium is shifted from all- trans isomers towards the 13-cis isomers. Two groups of values for calculated molar volume changes indicate that there are at least two different processes leading to a stable all-trans and 13-cis isomers' equilibrium called the dark-adapted bacteriorhodopsin. The first process may be attributed to changes in the distances and rearrangement of functionally important residues and a retinal Schiff base. It is suggested that the moved residues (probably Asp-212 with the contribution of Tyr-185 and/or Asp-85) closer to the chromophore could catalyse its trans-cis isomerization. These changes require smaller pressure changes and induce larger volume changes (large-volume-change process). The second process may be attributed to the formation of the three hydrogen bonds that additionally decrease the volume and strengthen further stabilization of the 13-cis isomer. To induce these changes, larger changes of pressure are required and the final molar volume changes are smaller (small-volume-change process). The total molar volume change between all-trans bacteriorhodopsin and 13-cis bacteriorhodopsin in the dark-adapted state of native bacteriorhodopsin was found to be about -28 mL/mol, which is much higher than the value of about -7 mL/mol obtained previously (Tsuda and Ebrey 1980, Schulte and Bradley 1995). The data provide a novel insight into factors leading to stable isomer equilibrium in dark-adapted bacteriorhodopsin.     

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.     

Spectrally silent transitions in the bacteriorhodopsin photocycle.

The photocycle kinetics of bacteriorhodopsin were analyzed from 0 to 40 degrees C at 101 wavelengths (330-730 nm). The data can be satisfactorily approximated by eight exponents. The slowest component (half-time 20 ms at 20 degrees C) belongs to the 13-cis cycle. The residual seven exponentials that are sufficient to describe the all-trans photocycle indicate that at least seven intermediates of the all-trans cycle must exist, although only five spectrally distinct species (K, L, M, N, and O) have been identified. These seven exponentials and their spectra at different temperatures provide the basis for the discussion of various kinetic schemes of the relaxation. The simplest model of irreversible sequential transitions includes after the first K--> L step the quasiequilibria of L<-->M, M<-->N, and N<-->O intermediates. These quasiequilibria are controlled by rate-limiting dynamics of the protein and/or proton transfer steps outside the chromophore region. Thus there exists an apparent kinetic paradox (i.e., why is the number of exponents of relaxation (at least seven) higher than the number of distinct spectral intermediates (only five)), which can be explained by assuming that some of the transitions correspond to changes in the quasiequilibria between spectrally distinct intermediates (i.e., are spectrally silent).     

Pathways of proton release in the bacteriorhodopsin photocycle.

The pH dependencies of the rate constants in the photocycles of recombinant D96N and D115N/D96N bacteriorhodopsins were determined from time-resolved difference spectra between 70 ns and 420 ms after photoexcitation. The results were consistent with the model suggested earlier for proteins containing D96N substitution: BR hv----K----L----M1----M2----BR. Only the M2----M1 back-reaction was pH-dependent: its rate increased with increasing [H+] between pH 5 and 8. We conclude from quantitative analysis of this pH dependency that its reverse, the M1----M2 reaction, is linked to the release of a proton from a group with a pKa = 5.8. This suggests a model for wild-type bacteriorhodopsin in which at pH greater than 5.8 the transported proton is released on the extracellular side from this as yet unknown group and on the 100-microseconds time scale, but at pH less than 5.8, the proton release occurs from another residue and later in the photocycle most likely directly from D85 during the O----BR reaction. We postulate, on the other hand, that proton uptake on the cytoplasmic side will be by D96 and during the N----O reaction regardless of pH. The proton kinetics as measured with indicator dyes confirmed the unique prediction of this model: at pH greater than 6, proton release preceded proton uptake, but at pH less than 6, the release was delayed until after the uptake. The results indicated further that the overall M1----M2 reaction includes a second kinetic step in addition to proton release; this is probably the earlier postulated extracellular-to-cytoplasmic reorientation switch in the proton pump.     

Proton transfers in the bacteriorhodopsin photocycle

The steps in the mechanism of proton transport in bacteriorhodopsin include examples for most kinds of proton transfer reactions that might occur in a transmembrane pump: proton transfer via a bridging water molecule, coupled protonation/deprotonation of two buried groups separated by a considerable distance, long-range proton migration over a hydrogen-bonded aqueous chain, and capture as well as release of protons at the membrane-water interface. The conceptual and technical advantages of this system have allowed close examination of many of these model reactions, some at an atomic level.     

On the photocycle and light adaptation of dark-adapted bacteriorhodopsin.

Pulsed Nd laser (25 ns, 530 nm) photolysis experiments were carried out at room temperature in aqueous suspensions of dark- and light-adapted fragments of the purple membrane of Halobacterium halobium. It is shown that the (50%) 13-cis isomeric component (BR13-cis) of dark-adapted bacteriorhodopsin (BRDA) undergoes a photocycle involving a characteristic transient absorbing in the neighborhood of 610 nm. At relatively high excitation intensities BR13-cis is converted to the same 410 nm (M) transient that characterized the photocycle of the all-trans isomer (BRtrans) of light-adapted bacteriorhodopsin (BRLA). This process, which competes with the generation of the "610" species, is attributed to the photo-induced conversion, during the pulse, of BR13-cis (or of its primary photoproduct "X") to a species in the BRtrans photocyte. The relationship between these observations and the mechanism of BRDA hv leads to BRLA adaptation at low excitation intensities (for which a quantum yield limit, 0 less than or equal to (3.5 +/- 0.7) X 10(-2) , is established) is discussed.