Infrared evidence that the Schiff base of bacteriorhodopsin is protonated: bR570 and K intermediates (original) (raw)
Related papers
Biochemistry, 1984
The first step of the bacteriorhodopsin (bR) photocycle involves the formation of a red-shifted product, K. Fourier transform infrared difference spectra of the bR570 to K630 transition at 81 K has been measured for bR containing different isotopic substitutions at the retinal Schiff base. In the case of bacteriorhodopsin containing a deuterium substitution at the Schiff base nitrogen, carbon 15, or both, we find spectral changes in the 1600-1610-and 1570-1580cm-' region consistent with the hypothesis that the K630 C=N stretching mode of a protonated Schiff base is located near 1609 cm-'. A similar set of Schiff base deuterium substitutions for retinal containing a I3C at the carbon 10 position strongly
Biochemistry, 1994
Bacteriorhodopsin contains all-trans-retinal linked via a protonated Schiff base to K2 16. The proton transport in this pump is initiated by all-trans to 13-cis photoisomerization of the retinal and the ensuing transfer of the Schiff base proton to D85. Changed geometrical relationship of the Schiff base and D85 after the photoisomerization is a possible reason for the proton transfer. We introduced small volume/shape changes with site-specific mutagenesis of residues V49 and A53 that contact the side chain of K216, in order to force the Schiff base into somewhat different positions relative to D85. Earlier [Ziminyi, L., Vir6, G., Chang, M., Ni, B., Needleman, R., & Lanyi, J. K. (1992) Biochemistry 31, 8535-85431 we had described the kinetics of absorbance changes in the microsecond to millisecond time range after photoexcitation with the scheme L -M I -M2 + H + (where the first equilibrium is the internal proton transfer and the second is proton release on the extracellular surface). Testing it at various p H values with mutants, where selected rate constants are changed, now confirms the validity of this scheme. The kinetics of the M state thus allowed examination of the transient equilibrium that develops in the L -M I reaction
Infrared study of the L, M, and N intermediates of bacteriorhodopsin using the photoreaction of M
Biochemistry, 1992
Infrared spectroscopy is used to characterize the transitions in the photocycle of bR involving the M intermediate. It has been shown previously that in this part of the photocycle a large protein conformational change takes place that is important for proton pumping. In this work we separate the spectra of the L, M, and N intermediates in order to better describe the timing of the molecular changes. We use the photoreaction of the M intermediate to separate its spectrum from those of L and N. At temperatures between 220 and 270 K a mixture of M and L or N is produced by illumination with green light. Subsequent blue illumination selectively drives M back into the ground state and the difference between the spectra before and after blue excitation yields the spectrum of M. Below about 250 K and L / M mixture is separated; at higher temperatures an M / N mixture is seen. We find that the spectrum of M is identical in the two temperature regions. The large protein conformational change is seen to occur during the M to N transition. Our results confirm that Asp-96 is transiently deprotonated in the L state. The only aspartic protonation changes between M and bR are the protonation of Asp-85 and Asp-21 2 that occur simultaneously during the L to M transition. Blue-light excitation of M results in deprotonation of both. The results suggest a quadrupolelike interaction of the Schiff base, Asp-85, Asp-21 2, and an additional positive charge in bR. Bacteriorhodopsin (bR) is a retinal-protein complex from the purple membrane patches in the cell membrane of Halobacterium halobium. Upon light absorption the light-adapted form bRLA pumps protons from the cell to the extracellular medium and the electrochemical energy of the resulting proton concentration difference is utilized by the cell. Light-induced proton pumping by bacteriorhodopsin is coupled to a series of successive reactions, the photocycle. Light absorption initiates a sequence of transitions in bR through metastable states (intermediates): a light-driven first step is followed by a series of thermally activated reactions, and the final state is the initial bR form. The following reaction scheme is regarded as an appropriate approximate description of this photocycle (Lozier et al., 1975
Fourier Transform Infrared Difference Spectroscopy of Bacteriorhodopsin and Its Photoproducts
Proceedings of The National Academy of Sciences, 1982
Fourier transform infrared difference spectroscopy has been used to obtain the vibrational modes. in the chro-*mophore and apoprotein 'that change in intensity or position between light-adapted bacteriorhodopsin and the K and M-intermediates in its photocycle and between dark-adapted and lightadapted bacteriorhodopsin. Our infrared' measurements provide independent verification of resonance Raman results that in lightadapted bacteriorhodopsin the protein-chromophore linkage is a protonated Schiff base and in the M state the Schiff base is un-,protonated. Although we cannot unambiguously identify the Schiff base stretching frequency in the K state, the most'likely interpretation of deuterium shifts of the chromophore hydrogen out-ofplane vibrations is that the Schiff base in K is protonated. The intensity of the hydrogen out-of-plane vibrations in the K state compared with the intensities of.those in light-adapted and'darkadapted bacteriorhodopsin shows that the conformation of the chromophore in K is considerably distorted. In addition, we find evidence that the conformation of the protein changes during the photocycle.
Structural Changes during the Formation of Early Intermediates in the Bacteriorhodopsin Photocycle
Biophysical Journal, 2002
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 NOH 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 NOH 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.
Photochemistry and photobiology, 1992
The photocycle of the proton pump bacteriorhodopsin contains two consecutive intermediates in which the retinal Schiff base is unprotonated; the reaction between these states, termed M1 and M2, was suggested to be the switch in the proton transport which reorients the Schiff base from D85 on the extracellular side to D96 on the cytoplasmic side (Váró and Lanyi, Biochemistry 30, 5016-5022, 1991). At pH 10 the absorption maxima of both M1 and M2 could be determined in the recombinant D96N protein. We find that M1 absorbs at 411 nm as do M1 and M2 in wild-type bacteriorhodopsin, but M2 absorbs at 404 nm. Thus, in M2 but not M1 the unprotonated Schiff base is affected by the D96N residue replacement. The connectivity of the Schiff base to D96 in the detected M2 state, but not in M1, is thereby established. On the other hand, the photostationary state which develops during illumination of D85N bacteriorhodopsin contains an M state corresponding to M1 with an absorption maximum shifted to...
On the molecular mechanisms of the Schiff base deprotonation during the bacteriorhodopsin photocycle
Proceedings of the National Academy of Sciences, 1986
Using optical flash photolysis and timeresolved Raman methods, we examined intermediates formed during the photocycle of bacteriorhodopsin (bR), as well as the bR color change, as a function of pH (in the 7.0-1.5 region) and as a function of the number of bound Ca2+ ions. It is found that at a pH just below 3 or with less than two bound Ca21 per bR, the deprotonation (the L550 -_ M412) step ceases, yet the K610 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.