Using time-resolved step-scan FTIR spectroscopy, it has become possible to identify two different M states in the 5 to 300 μs time range, which clearly differ from the M N state. The identification has become possible by measuring a pure BR→KL difference spectrum at 100 ns, which can be used to correct for the contributions of this intermediate in the later difference spectra. The subtraction of the later corrected difference spectra thus represent L→M 1 , M 2 difference spectra with varying relative amount of the two M states. The comparison of these spectra clearly reveals differences in the amide-II spectral range, and possibly also in the amide-I range. From these observations it can be concluded that the two M states do not differ in the chromophore structure but in the protein structure.

In this report, from time-resolved step-scan Fourier transform infrared investigations from 15ns to 160ms, we provide evidence for the subsequent rise of three different M states that differ in their structures. The first state rises with ∼3μs to only a small percentage. Its structure as judged from amide I/II bands differs in small but well-defined aspects from the L state. The next M state, which appears in ∼40μs, has almost all of the characteristics of the “late” M state, i.e., it differs considerably from the first one. Here, the L ↔ M equilibrium is shifted toward M, although some percentage of L still persists. In the last M state (rise time ∼130μs), the equilibrium is shifted toward full deprotonation of the Schiff base, and only small additional structural changes take place. In addition to these results obtained for unbuffered conditions or at pH 7, experiments performed at lower and higher pH are presented. These results are discussed in terms of the molecular changes postulated to occur in the M intermediate to allow the shift of the L/M equilibrium toward M and possibly to regulate the change of the accessibility of the Schiff base necessary for effective proton pumping.

Our improved step-scan FTIR instrument, capable of measuring spectra within 15 ns after the flash, is employed to measure flash-induced infrared difference spectra of bacteriorhodopsin, halorhodopsin and CO-myoglobin. For all three systems it is necessary to cover a large time range extending into several milliseconds. Therefore, the linear time base provided by the transient recorder board is converted to a quasi-logarithmic scale. Each of the three systems is characterized by several time constants extending over the large time range. For bacteriorhodopsin, it is shown that two spectral changes occur, one in the 20 and the other in the 100 ns time range. Furthermore, spectral differences between the two M states could be detected in the mu s time range. For halorhodopsin, a clear bathe intermediate with red-shifted ethylenic mode could be identified in the nanosecond time range. In addition, a transition corresponding to the N intermediate in bacteriorhodopsin was deduced. Further, it is shown that the millisecond time constant depends on Cl- concentration, enabling the detection of the O intermediate. In the case of CO-myoglobin, spectral differences could be identified caused by mutations of the distal histidine of the heme binding pocket.

Time-resolved vibrational spectra are used to elucidate the structural changes in the retinal chromophore within the K-590 intermediate that precedes the formation of the L-550 intermediate in the room-temperature (RT) bacteriorhodopsin (BR) photocycle. Measured by picosecond time-resolved coherent anti-Stokes Raman scattering (PTR/CARS), these vibrational data are recorded within the 750 cm-1 to 1720 cm-1 spectral region and with time delays of 50-260 ns after the RT/BR photocycle is optically initiated by pulsed (< 3 ps, 1.75 nJ) excitation. Although K-590 remains structurally unchanged throughout the 50-ps to 1-ns time interval, distinct structural changes do appear over the 1-ns to 260-ns period. Specifically, comparisons of the 50-ps PTR/CARS spectra with those recorded with time delays of 1 ns to 260 ns reveal 1) three types of changes in the hydrogen-out-of-plane (HOOP) region: the appearance of a strong, new feature at 984 cm-1; intensity decreases for the bands at 957 cm-1, 952 cm-1, and 939 cm-1; and small changes intensity and/or frequency of bands at 855 cm-1 and 805 cm-1; and 2) two types of changes in the C-C stretching region: the intensity increase in the band at 1196 cm-1 and small intensity changes and/or frequency shifts for bands at 1300 cm-1 and 1362 cm-1. No changes are observed in the C = C stretching region, and no bands assignable to the Schiff base stretching mode (C = NH+) mode are found in any of the PTR/CARS spectra assignable to K-590. These PTR/CARS data are used, together with vibrational mode assignments derived from previous work, to characterize the retinal structural changes in K-590 as it evolves from its 3.5-ps formation (ps/K-590) through the nanosecond time regime (ns/K-590) that precedes the formation of L-550. The PTR/CARS data suggest that changes in the torsional modes near the C14-C15 = N bonds are directly associated with the appearance of ns/K-590, and perhaps with the KL intermediate proposed in earlier studies. These vibrational data can be primarily interpreted in terms of the degree of twisting of the C14-C15 retinal bond. Such twisting may be accompanied by changes in the adjacent protein. Other smaller, but nonetheless clear, spectral changes indicate that alterations along the retinal polyene chain also occur. The changes in the retinal structure are preliminary to the deprotonation of the Schiff base nitrogen during the formation of M-412. The time constant for the ps/ns K-590 transformation is estimated from the amplitude change of four vibrational bands in the HOOP region to be 40-70 ns.

Vibrational spectra of bacteriorhodopsin (BR) in its deionized form (blue-membrane BR) were measured via picosecond resonance coherent anti-Stokes Raman scattering (PR/CARS). More than 18 vibrational bands in the 750–1650 cm-1 region not previously assigned were observed. Comparisons of these PR/CARS spectra with vibrational data recorded from BR species containing either an all-trans (i.e. BR-570) or 13-cis retinal (e.g. BR-548 found in dark-adapted BR and the picosecond BR intermediate K-590) were used to characterise more fully isomeric forms of the retinal chromophore found in blue-membrane BR. © 1997 by John Wiley & Sons, Ltd.

The hypothesis was tested whether in bacteriorhodopsin (BR) the reduction of the steric interaction between the 9-methyl group of the chromophore all-trans-retinal and the tryptophan at position 182 causes the same changes as observed in the photocycle of 9-demethyl-BR. For this, the photocycle of the mutant W182F was investigated by time-resolved UV-vis and pH measurements and by static and time-resolved FT-IR difference spectroscopy. We found that the second half of the photocycle was similarly distorted in the two modified systems: based on the amide-I band, the protonation state of D96, and the kinetics of proton uptake, four N intermediates could be identified, the last one having a lifetime of several seconds; no O intermediate could be detected; the proton uptake showed a pronounced biphasic time course; and the pKa of group(s) on the cytoplasmic side in N was reduced from 11 in wild type BR to around 7.5. In contrast to 9-demethyl-BR, in the W182F mutant the first part of the photocycle does not drastically deviate from that of wild type BR. The results demonstrate the importance of the steric interaction between W182 and the 9-methyl group of the retinal in providing tight coupling between chromophore isomerization and the late proton transfer steps.

The photocycle of bacteriorhodopsin (BR) regenerated with all-trans-9-demethylretinal was investigated by time-resolved rapid-scan Fourier transform infrared difference spectroscopy, by static low-temperature difference spectroscopy at 80, 170, and 213 K and by static steady-state difference spectroscopy at 278 K. In addition, the formation and decay of M intermediate was monitored at 412 nm with conventional flash photolysis experiments. Our data show that the removal of the 9-methyl group strongly changes the photocycle of BR. The reaction cycle is slowed down about 250-fold. The photoreaction is characterized by a slow rise of the M intermediate and by a very long-lived N intermediate. No O intermediate could be observed. The low-temperature spectra indicate that already at 80 K a KL-like photoproduct is formed. L can be obtained as in native BR at 170 K, but its decay appears to be inhibited, since it can still be observed at 213 K and high pH, in addition to the M intermediate. As in native BR, the 15-hydrogen out-of-plane modes of the L and N intermediates (observed in 2H2O) are very similar. Evidence for the existence of three N substates which differ in the protonation state of Asp96 and in the amide I bands is presented. This is explained by the extremely slowed-down reisomerization of the chromophore. The results are discussed with respect to alterations in the chromophore-protein interaction, caused by the removal of the 9-methyl group.

The recombination of Myoglobin (Mb) and CO after laser flash induced photolysis of Carboxymyoglobin (MbCO) in aqueous solution has been studied by Time-resolved Fourier-Transform Infrared Spectroscopy (FTIR) using a Step-Scan-Interferometer. Difference specra in the spectral range between 1100 cm−1 and 2100 cm−1 were obtained at different times after photodissociation. The kinetics of the CO stretching band and the amide-II band show effects of photoselection.

Sub-microsecond time-resolved step-scan FT-IR spectroscopy is applied to the study of the molecular changes and their dynamics occurring during the KL-L transition of bacteriorhodopsin. The time-resolved difference spectra are compared to the static low-temperature BR → K and BR → L difference spectra. Our data show that the protein part in KL is similar to that in K. However, the chromophore is more relaxed and is differently twisted. A strong hydrogen-out-of-plane (HOOP) mode in KL is assigned to the 15-HOOP. As is the case for L, a strong deformation of the C 14 -C 15 single bond is deduced for KL. Evidence of a KL → L equilibrium is presented. In N, a 15-HOOP mode similar to that in L is observed, indicating very similar twists of the C 14 -C 15 single bond. This observation excludes major contributions of this deformation to the reduction of the pK a of the Schiff base in L. From the spectral changes, important molecular events are deduced that occur in the transitions to KL, L, and N.

Purple membrane was regenerated from the denatured proteolytic (protease V8) fragments V-1 and V-2 of bacteriorhodopsin (BR), native membrane lipids, and all-trans-retinal. FTIR difference spectra of M and N intermediates of the reconstituted system are in close correspondence to those obtained from native BR. Asp-212 is the only internal aspartic acid in the V-2 fragment (helices F and G). Reconstituting a V-2 fragment from a [4-13C]Asp-labeled BR preparation with an unmodified V-1 fragment and vice versa have allowed us to assign IR bands to either Asp-212 or any of the remaining aspartic acids on V-1 (helices A-E). A carboxylate vibration at 1392 cm-1 has been identified in the M and N intermediates and assigned to Asp-212. Since no contribution of this residue to C = O stretches of protonated carboxyl groups was detected, Asp-212 must be ionized in light-adapted BR as well. The effect of [4-13C]Asp labeling of V-1 revealed a carboxylate vibration at 1385 cm-1 in light-adapted BR. Since Asp-96 and Asp-115 are protonated, this band is caused by Asp-85. All absorption changes of C = O stretches of protonated carboxyl groups are due to Asp residues on V-1. Correspondingly, the proton acceptor for Schiff base deprotonation in M is located on V-1, and must be Asp-85 (the only ionized Asp on V-1). The band assignments are compared with those reported for BR mutants, and the potential role of Asp-212 for proton translocation is discussed.

Time-resolved IR difference spectroscopy is an ideal tool to study molecular processes in photobiological systems, since it is sensitive to both chromophore and protein alterations. Recently, a new powerful method, time-resolved FT-IR absorption spectroscopy using a step-scan interferometer, has been described [1]. The time-resolution has now been increased to ca. 200 ns, and the accuracy for the slower processes has been increased by the use of a quasi-logarithmic transient-recorder. In this report, this method is applied to the study of the BR-gt K (KL) transition and to the molecular discrimination of the M and N intermediates. In Fig. 1, the time-resoved (500 ns after the flash) and the static (temperature 80 K) BR-gt K difference spectra are compared. The overall agreement is surprisingly good. It is noteworthy that the same spectral features in the amide-I range are observed in both spectra, indicating comparable protein structural changes at 80 K vs. 500 ns after the flash at room temperature. However, the small band at 1555 cm−1, present in the low-temperature spectrum, is missing in the time-resolved studies. The main difference, however, can be seen in the HOOP spectral range below 1000 cm−1: whereas in the low-temperature spectrum a HOOP mode at 955 cm−1 (11,12-HOOP) dominates, in the room-temperature spectrum a mode at 980 cm−1, which is absent in the former spectrum, exhibits the largest intensity.

Abstract— In order to assign the proton acceptor for Schiff base deprotonation in bacteriorhodopsin to a specific Asp residue, the photoreaction of the Asp85 → Glu mutant, as expressed in Halobacterium sp. GRB, was investigated by static low‐temperature and time‐resolved infrared difference spec‐troscopy. Measurements were also performed on the mutant protein labeled with [4‐13C]Asp which allowed discrimination between Asp and Glu residues. 14,15‐di13C‐retinal was incorporated to distinguish amide‐II absorbance changes from changes of the ethylenic mode of the chromophore. In agreement with earlier UV‐VIS measurements, our data show that from both the 540 and 610 nm species present in a pH‐dependent equilibrium, intermediates similar to K and L can be formed. The 14 ms time‐resolved spectrum of the 540 nm species shows that a glutamic acid becomes protonated in the M‐like intermediate, whereas the comparable difference spectrum of the 610 nm species demonstrates that in the initial state a glutamic acid is already protonated. In conjunction with earlier observations of protonation of an Asp residue in wild‐type M, the data provide direct evidence that the proton acceptor in the deprotonation reaction of the Schiff base is Asp85.