Chromophore motion during the bacteriorhodopsin photocycle: polarized absorption spectroscopy of bacteriorhodopsin and its M-state in bacteriorhodopsin crystals.

The three-dimensional crystallization of bacteriorhodopsin was systematically investigated and the needle-shaped crystal form analysed. In these crystals the M-intermediate forms 10 times faster and decays 15 times more slowly than in purple membranes. Polarized absorption spectra of the crystals were measured in the dark and light adapted states. A slight decrease in the angle between the transition moment and the membrane plane was detected during dark adaptation. The crystallization of a mutated bacteriorhodopsin, in which the aspartic acid at residue 96 was replaced by asparagine, provided crystals with a long lived M-intermediate. This allowed polarized absorption measurements of the M-chromophore. The change in the polarization ratio upon formation of the M-intermediate indicates an increase in the angle between the main transition dipole and the membrane plane by 2.2 degrees +/- 0.5, corresponding to a 0.5 A displacement of one end of the chromophore out of the membrane plane of the bacteriorhodopsin molecule.     

Photolytic interruptions of the bacteriorhodopsin photocycle examined by time-resolved resonance raman spectroscopy

An investigation of the photolytic conditions used to initiate and spectroscopically monitor the bacteriorhodopsin (BR) photocycle utilizing time-resolved resonance Raman (TR3) spectroscopy has revealed and characterized two photoinduced reactions that interrupt the thermal pathway. One reaction involves the photolytic interconversion of M-412 and M', and the other involves the direct photolytic conversion of the BR-570/K-590 photostationary mixture either to M-412 and M' or to M-like intermediates within 10 ns. The photolytic threshold conditions describing both reactions have been quantitatively measured and are discussed in terms of experimental parameters.     

Photoacoustic spectroscopy of purple membrane fragments. Evidence for disorder during photocycle

A strong band at 412 nm has been observed in the photoacoustic spectrum of partially dried purple membrane, peaking sharply at a modulation frequency of about 70 Hz. This may be explained in terms of a disorder-order transition.     

Picosecond time-resolved fluorescence spectroscopy of K-590 in the bacteriorhodopsin photocycle.

The fluorescence spectrum of a distinct isometric and conformational intermediate formed on the 10(-11) s time scale during the bacteriorhodopsin (BR) photocycle is observed at room temperature using a two laser, pump-probe technique with picosecond time resolution. The BR photocycle is initiated by pulsed (8 ps) excitation at 565 nm, whereas the fluorescence is generated by 4-ps laser pulses at 590 nm. The unstructured fluorescence extends from 650 to 880 nm and appears in the same general spectral region as the fluorescence spectrum assigned to BR-570. The transient fluorescence spectrum can be distinguished from that assigned to BR-570 by a larger emission quantum yield (approximately twice that of BR-570) and by a maximum intensity near 731 nm (shifted 17 nm to higher energy from the maximum of the BR-570 fluorescence spectrum). The fluorescence spectrum of BR-570 only is measured with low energy, picosecond pulsed excitation at 590 nm and is in good agreement with recent data in the literature. The assignment of the transient fluorescence spectrum to the K-590 intermediate is based on its appearance at time delays longer than 40 ps. The K-590 fluorescence spectrum remains unchanged over the entire 40-100-ps interval. The relevance of these fluorescence data with respect to the molecular mechanism used to model the primary processes in the BR photocycle also is discussed.     

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.     

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.     

Nanosecond time-resolved infrared spectroscopy distinguishes two K species in the bacteriorhodopsin photocycle.

The photochemical reaction process of bacteriorhodopsin in the nanosecond time range (-120-860 ns) was measured in the 1400-900 cm-1 region with an improved time resolved dispersive-type infrared spectrometer. The system is equipped with a newly developed detection unit whose instrumental response to a 5-ns laser pulse has a full width of the half-maximum of 60 ns. It provides highly accurate data that enabled us to extract a kinetic process one order of magnitude faster than the instrumental response. The spectral changes in the 1400-900 cm-1 region were analyzed by singular value decomposition and resolved into three components. These components were separated by fitting with 10- and 1000-ns exponential functions and a step function, which were convoluted with the instrumental response function. The components with decay time constants of 10 and 1000 ns are named K and KL, respectively, on the basis of previous visible spectroscopy. The spectral shapes of K and KL are distinguishable by their hydrogen-out-of-plane (HOOP) modes, at 958 and 984 cm-1, respectively. The former corresponds to the K intermediate recorded at 77 K and the latter to a K-like photoproduct at 135 K. On the basis of published data, these bands are assigned to the 15-HOOP mode, indicating that the K and KL differ in a twist around the C14-C15 bond.     

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.     

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.     

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.     

Water structural changes in the bacteriorhodopsin photocycle: analysis by Fourier transform infrared spectroscopy.

The Fourier transform infrared difference spectra between light-adapted bacteriorhodopsin (BR) and its photointermediates, L and M, were analyzed for the 3750-3450-cm-1 region. The O-H stretching vibrational bands were identified from spectra upon substitution with 2H2O. Among them, the 3642-cm-1 band of BR was assigned to water by substitution with H2(18)O. By a comparison with the published infrared spectra of the water in model systems [Mohr, S.C., Wilk, W.D., & Barrow, G.M. (1965) J. Am. Chem. Soc. 87, 3048-3052], it is shown that the O-H bonds of the water in BR interact very weakly. Upon formation of L, the interaction becomes stronger. The O-H bonds of the protein side chain undergo similar changes. On the other hand, M formation further weakens the interaction of the same water molecules in BR. The appearance of a sharp band at 3486 cm-1, which was assigned tentatively to the N-H stretching vibration of the peptide bond, is unique to L. The results suggest that the water molecules are involved in the perturbation of Asp-96 in the L intermediate and that they are exerted from the protonated Schiff base which changes position upon the light-induced reaction.     

Effect of cross linkers on the bacteriorhodopsin photocycle

A general behavior of bacteriorhodopsin in purple membranes from Halobacterium halobium has been observed upon modification resulting in cross-linking of carboxyl and lysine groups. The rise of the M-intermediate contained two components with approximately 50-50% intensity; its decay showed three components with approximately 25-50-25% intensity respectively in a pH range of 5-9. The significance of these remarkably similar data with respect to the proton translocation mechanism in bacteriorhodopsin is that chemical modification allows us to conclude that disturbing parts of the hypothetical "proton conducting chain" does not inhibit proton translocation.     

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.     

Vibrational spectroscopy of bacteriorhodopsin mutants: I. Tyrosine-185 protonates and deprotonates during the photocycle

The techniques of FTIR difference spectroscopy and site-directed mutagenesis have been combined to investigate the role of individual tyrosine side chains in the proton-pumping mechanism of bacteriorhodopsin (bR). For each of the 11 possible bR mutants containing a single Tyr----Phe substitution, difference spectra have been obtained for the bR----K and bR----M photoreactions. Only the Tyr-185----Phe mutation results in the disappearance of a set of bands that were previously shown to be due to the protonation of a tyrosinate during the bR----K photoreaction [Rothschild et al.: Proceedings of the National Academy of Sciences of the United States of America 83:347, (1986]). The Tyr-185----Phe mutation also eliminates a set of bands in the bR----M difference spectrum associated with deprotonation of a Tyr; most of these bands (e.g., positive 1272-cm-1 peak) are completely unaffected by the other ten Tyr----Phe mutations. Thus, tyrosinate-185 gains a proton during the bR----K reaction and loses it again when M is formed. Our FTIR spectra also provide evidence that Tyr-185 interacts with the protonated Schiff base linkage of the retinal chromophore, since the negative C = NH+ stretch band shifts from 1640 cm-1 in the wild type to 1636 cm-1 in the Tyr-185----Phe mutant. A model that is consistent with these results is that Tyr-185 is normally ionized and serves as a counter-ion to the protonated Schiff base. The primary photoisomerization of the chromophore translocates the Schiff base away from Tyr-185, which raises the pKa of the latter group and results in its protonation.     

Time-resolved refractive index change during the bacteriorhodopsin photocycle

Kinetic refractive index spectroscopy has been applied to the study of the bacteriorhodopsin photocycle. A fully hydrated purple membrane film was examined in the temperature range from 10° to 40°C using 532 nm excitation (doubled Nd YAG laser) and 633 nm (He–Ne laser) testing beam. Multiexponential fitting of the data revealed five processes. Four of them are well known from kinetic optical absorption studies. The fifth process has only recently been observed in optical absorption experiments where it has a relatively small amplitude. In our refractive index experiments it has an amplitude of up to 30% of the full signal amplitude. It is characterized by an Arrhenius temperature dependence with an activation enthalpy of 40±5 kJ/mol and a decay time of about 0.8 ms at 20°C.     

Thermodynamics and energy coupling in the bacteriorhodopsin photocycle

Time-resolved absorption changes of photoexcited bacteriorhodopsin were measured with a gated multichannel analyzer between 100 ns and 100 ms at six temperatures between 5 and 30 degrees C. The energetics of the chromophore reaction cycle were analyzed on the basis of a model containing a single cycle and reversible reactions. The calculated thermodynamic parameters provide insights to general principles of the active transport. They indicate that in this light-driven proton pump the free energy is retained after absorption of the photon as the enthalpy of the pKa shift in the chromophore which allows deprotonation of the Schiff base. Part of the excess free energy is dissipated at the "switch" step where the reaction and transport cycles are coupled, and the rest at the chromophore recovery step. All other reactions take place near equilibrium. The "switch" step is the M1----M2 transition in the reaction cycle [Váró, G., & Lanyi, J. K. (1991) Biochemistry (preceeding paper in this issue)]. It provides for return of the chromophore pKa to its initial value so the Schiff base will become a proton acceptor, for reordering access of the Schiff base from one side of the membrane to the other, and for unidirectionality of the proton transfer. Conformational energy of the protein, acquired during the "switch" step, drives the completion of the photocycle.