Structural changes during the formation of early intermediates in the bacteriorhodopsin photocycle

Early intermediates of bacteriorhodopsin's photocycle were modeled by means of ab initio quantum mechanical/molecular mechanical and molecular dynamics simulations. The photoisomerization of the retinal chromophore and the formation of photoproducts corresponding to the early intermediates were simulated by molecular dynamics simulations. By means of the quantum mechanical/molecular mechanical method, the resulting structures were refined and the respective excitation energies were calculated. Two sequential intermediates were found with absorption maxima that exhibit red shifts from the resting state. The intermediates were therefore assigned to the K and KL states. In K, the conformation of the retinal chromophore is strongly deformed, and the N--H bond of the Schiff base points almost perpendicular to the membrane normal toward Asp-212. The strongly deformed conformation of the chromophore and weakened interaction of the Schiff base with the surrounding polar groups are the means by which the absorbed energy is stored. During the K-to-KL transition, the chromophore undergoes further conformational changes that result in the formation of a hydrogen bond between the N--H group of the Schiff base and Thr-89 as well as other rearrangements of the hydrogen-bond network in the vicinity of the Schiff base, which are suggested to play a key role in the proton transfer process in the later phase of the photocycle.     

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.     

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.     

Photoacoustic spectroscopy of bacteriorhodopsin photocycle

Photoacoustic spectroscopy was applied to study the energetics and the kinetics of the slow intermediates of the bacteriorhodopsin photocycle. An analysis of the modulation frequency dependence of the photoacoustic signal allowed us to estimate the enthalpy changes and the kinetic parameters associated with those intermediates. The effects of pH, salt concentration, and protein aggregation were studied. Three photoacoustic transitions were found. The two low frequency transitions were attributed to O660 and M412, respectively. The third transition was interpreted as resulting from a protein conformational change undetected spectrophotometrically. The frequency spectra were simulated between 5 and 180 Hz at pH's 5.1, 7.0, and 8.9 assuming a branching in the bacteriorhodopsin photocycle at the M412 level. The enthalpy changes associated with M412 and O660 were computed and compared with the experimental values.     

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.     

Interconversions among four M-intermediates in the bacteriorhodopsin photocycle.

Halobacterium salinarum displays four distinct kinetic forms of M-intermediate in its bacteriorhodopsin photocycle. In wild-type, there are mainly two species with time constants near 2 and 5 ms. Under various kinds of stress, two other species arise with time constants near 10 and 70 ms. We show that these four species are interconvertible. Increases in membrane hydrophobicity convert the slower to faster forms. Perturbations caused by Triton X-100 or mutations convert faster to slower forms. The fastest form requires a hydrophobic membrane environment near a ring of four charged aspartate residues in the trimer, namely Asp36, Asp38, Asp102, and Asp104 in the cytoplasmic loop regions. Interconversions of the 2-ms and 5-ms species of the wild-type are accomplished by pH-changes. The potential significance of these findings is discussed.     

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.     

Distortions in the photocycle of bacteriorhodopsin at moderate dehydration.

The photoreaction of bacteriorhodopsin was studied in moderately dehydrated films (relative humidities between 100 and 65%). Time-resolved difference spectra from a gated optical multichannel analyzer, between 100 ns and 100 ms after photoexcitation, were decomposed into sums of difference spectra of the intermediates K, L, M, N, and O, and the kinetics obtained were fitted to various alternative schemes. The data confirm the model of a single reaction sequence with reversible reactions we proposed recently for purple membrane suspensions (Váró, G., and J. K. Lanyi. Biochemistry. 1990. 29:2241-2250) but including reversibility also for the reaction K in equilibrium with L in addition to L in equilibrium with M, M in equilibrium with N, and N in equilibrium with O. With increasing dehydration the kinetics were increasingly dominated by the reverse reactions. As before, fitting the data required the existence of two M species in series: L in equilibrium with M1 in equilibrium with M2 in equilibrium with N. The M1 in equilibrium with M2 reaction was greatly slowed at lower humidities. This step might be the switch for the unidirectional transfer of protons. With increasing dehydration recovery of BR occurred less and less via the N intermediate and increasingly via direct shunts from the two M species. As indicated earlier by electrical measurements with similarly dried bacteriorhodopsin films (Váró, G., and L. Keszthelyi, 1983. Biophys. J. 43:47-51). The latter are pathways not necessarily associated with net proton translocation.     

Structural changes in bacteriorhodopsin during the photocycle measured by time-resolved polarized Fourier transform infrared spectroscopy.

The structural changes in bacteriorhodopsin during the photocycle are investigated. Time resolved polarized infrared spectroscopy in combination with photoselection is used to determine the orientation and motion of certain structural units of the molecule: Asp-85, Asp-96, Asp-115, the Schiff base, and several amide I vibrations. The results are compared with recently published x-ray diffraction data with atomic resolution about conformational motions during the photocycle. The orientation of the measured vibrations are also calculated from the structure data, and based on the comparison of the values from the two techniques new information is obtained: several amide I bands in the infrared spectrum are assigned, and we can also identify the position of the proton in the protonated Asp residues.     

On the primary quantum yields in the bacteriorhodopsin photocycle.

Pulsed Nd laser experiments in suspensions of the purple membrane of Halobacterium halobium are carried out at room temperature. At sufficiently high laser intensities, a photostationary mixture of bacteriorhodopsin (BR) and its red-shifted (batho) photoproduct (K) is obtained. The spectra of the first three intermediates in the photocycle are reported. The data yield a value of phi1/phi2=0.40 +/- 0.05 for the ratio of the quantum yields of the forward (phi1) and reverse (phi2) processes, setting an upper limit of approximately 0.4 for the quantum efficiency of the cycle at room temperature. This method is generally available for the determination of phi2 in the case of a photoequilibrium: A in equilibrium B, where B is a short-lived transient and phi1 is known from low intensity measurements. Its potential application is of importance for the study of the photophysics of visual pigments at physiological temperatures.     

X-ray diffraction of bacteriorhodopsin photocycle intermediates

Recent advances in the crystallography of bacteriorhodopsin, the light-driven proton pump, have yielded structural models for all intermediates of the photochemical cycle. For seven of the species, X-ray diffraction data were collected from trapped photostationary states in crystals, and for the two remaining ones the structures of selected mutants are available. The changes of the retinal chromophore, protein and bound water describe, at an atomic level, how accommodation of the twisted photoisomerized retinal to its binding site causes de-protonation of the retinal Schiff base and initiates cascades of gradual conformational rearrangements of the protein. One cascade propagates in the extracellular direction and results in proton release, and the other in the cytoplasmic direction and results in side-chain and main-chain rearrangements, formation of a chain of hydrogen-bonded water, and proton uptake from the bulk. Such local-global conformational coupling, with gradual spreading of a local perturbation over the rest of the protein, might be the uniting principle of transporters and receptors.     

Protein conformational changes in the bacteriorhodopsin photocycle

We report a comprehensive electron crystallographic analysis of conformational changes in the photocycle of wild-type bacteriorhodopsin and in a variety of mutant proteins with kinetic defects in the photocycle. Specific intermediates that accumulate in the late stages of the photocycle of wild-type bacteriorhodopsin, the single mutants D38R, D96N, D96G, T46V, L93A and F219L, and the triple mutant D96G/F171C/F219L were trapped by freezing two-dimensional crystals in liquid ethane at varying times after illumination with a light flash. Electron diffraction patterns recorded from these crystals were used to construct projection difference Fourier maps at 3.5 A resolution to define light-driven changes in protein conformation.Our experiments demonstrate that in wild-type bacteriorhodopsin, a large protein conformational change occurs within approximately 1 ms after illumination. Analysis of structural changes in wild-type and mutant bacteriorhodopsins under conditions when either the M or the N intermediate is preferentially accumulated reveals that there are only small differences in structure between M and N intermediates trapped in the same protein. However, a considerably larger variation is observed when the same optical intermediate is trapped in different mutants. In some of the mutants, a partial conformational change is present even prior to illumination, with additional changes occurring upon illumination. Selected mutations, such as those in the D96G/F171C/F219L triple mutant, can sufficiently destabilize the wild-type structure to generate almost the full extent of the conformational change in the dark, with minimal additional light-induced changes. We conclude that the differences in structural changes observed in mutants that display long-lived M, N or O intermediates are best described as variations of one fundamental type of conformational change, rather than representing structural changes that are unique to the optical intermediate that is accumulated. Our observations thus support a simplified view of the photocycle of wild-type bacteriorhodopsin in which the structures of the initial state and the early intermediates (K, L and M1) are well approximated by one protein conformation, while the structures of the later intermediates (M2, N and O) are well approximated by the other protein conformation. We propose that in wild-type bacteriorhodopsin and in most mutants, this conformational change between the M1 and M2 states is likely to make an important contribution towards efficiently switching proton accessibility of the Schiff base from the extracellular side to the cytoplasmic side of the membrane.     

Pressure dependence of the photocycle kinetics of bacteriorhodopsin

The pressure dependence of the photocycle kinetics of bacteriorhodopsin from Halobacterium salinarium was investigated at pressures up to 4 kbar at 25 degrees C and 40 degrees C. The kinetics can be adequately modeled by nine apparent rate constants, which are assigned to irreversible transitions of a single relaxation chain of nine kinetically distinguishable states P(1) to P(9). All states except P(1) and P(9) consist of two or more spectral components. The kinetic states P(2) to P(6) comprise only the two fast equilibrating spectral states L and M. From the pressure dependence, the volume differences DeltaV(o)(LM) between these two spectral states could be determined that range from DeltaV(o)(LM) = -11.4 +/- 0.7 ml/mol (P(2)) to DeltaV(o)(LM) = 14.6 +/- 2.8 mL/mol (P(6)). A model is developed that explains the dependence of DeltaV(o)(LM) on the kinetic state by the electrostriction effect of charges, which are formed and neutralized during the L/M transition.     

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.