The molecular changes during the photoreaction of halorhodopsin from Natronobacterium pharaonis have been monitored by low-temperature static and by time-resolved step-scan Fourier transform infrared difference spectroscopy. In the low-temperature L spectrum anions only influence a band around 1650 cm(-1), tentatively assigned to the C=N stretch of the protonated Schiff base of L. The analysis of the time-resolved spectra allows to identify the four states: K, L-1, L-2, and O. Between L-1 and L-2, only the apoprotein undergoes alterations. The O state is characterized by an all-trans chromophore and by rather large amide I spectral changes. Because in our analysis the intermediate containing O is in equilibrium with a state indistinguishable from L-2, we are unable to identify an N-like state. At very high chloride concentrations (>5 M), we observe a branching of the photocycle from L-2 directly back to the dark state, and we provide evidence for direct back-isomerization from L-2. This branching leads to the reported reduction of transport activity at such high chloride concentrations. We interpret the L-1 to L-2 transition as an accessibility change of the anion from the extracellular to the cytosolic side, and the large amide I bands in O as an indication for opening of the cytosolic channel from the Schiff base toward the cytosolic surface and/or as indication for changes of the binding constant of the release site.

The implementation of a sample changing wheel in combination with a step-scan FTIR spectrometer is described. This device allows collecting and averaging time-resolved data of ten samples, which can be placed successively into the infrared beam. The high time-resolution of the step-scan technique can be retained to monitor the fast kinetics of systems with slow reaction cycles. Averaging the signals of several samples instead of signals of only one sample allows collecting the data with a repetition rate larger than what would be allowed by the time constant of the reaction cycle. Thus, instrumental instabilities due to a very long measuring time are avoided. It is demonstrated that, if at each mirror position the signals of the same samples are averaged, no multiplicative noise is introduced into the spectra. Results on the application of our method to the photocycle of the Asp96→Asn mutant of bacteriorhodopsin, which is slowed down by a factor of more than hundred compared to the native protein, are presented.

Several sources of errors leading to distortions in time-resolved step-scan Fourier transform infrared (FT-IR) measurements are described, and their effect on difference spectra and time traces is discussed. Fluctuations of the movable interferometer mirror as well as interference of the power line frequency are shown to be able to cause oscillations in the time traces of absorbance changes obtained by step-scan measurements. It is demonstrated that absorbance changes of the sample and thermal IR signals due to heating of the sample by laser excitation both contribute to the IR intensity changes at a fixed sampling position. Therefore, intensity variations of the laser pulses used to drive the sample reaction can cause considerable fluctuations of the time-dependent IR signals and, thereby, introduce multiplicative noise into the difference spectra. Means to correct these errors are proposed for some cases.

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.

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.

Replacing the chromophore of bacteriorhodopsin with chemically modified retinal analogs which cannot isomerize, allows to measure directly side effects from the laser pulse used for sample excitation in step-scan FT-IR measurements. Comparison with static temperature difference spectra and temperature-jump experiments shows that the observed effects can mainly be attributed to heating of the sample by the laser pulse. The size of resulting spectral changes is compared to difference bands of the photoreaction.

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.

A broadband amplifer with a short rise time was developed for the acquisition of the time dependent part of the interferogram in time-resolved step-scan measurements. Amplification of this portion of the interferogram by about 40 dB is necessary to digitize siganl and noise with adequate amplitude resolution. Therefore, the transient ac-signal from the detector has to be separated from the static de-signal being larger by a factor of thousand. We describe here a way to avoid the currently used ac-coupling of the signal which is always related to the disadvantage of a low frequency limit for time-dependent signals. Since the recorded interferogram shows distorsions caused by nonlinear response of the MCT-detector, especially, when a photoconductive detector is used, we implemented a software noninearity correction method proposed by Keens and Simon (1). The effects of the nolinearity correction on the time-resolved step-scan difference spectra of biological samples are demonstrated.

A broadband amplifier with a short rise time was developed for the acquisition of the time dependent part of the interferogram in time-resolved step-scan measurements. Amplification of this portion of the interferogram by about 40 dB is necessary to digitize signal and noise with adequate amplitude resolution. Therefore, the transient ac-signal from the detector has to be separated from the static dc-signal being larger by a factor of thousand. We describe here a way to avoid the currently used ac-coupling of the signal which is always related to the disadvantage of a low frequency limit for time-dependent signals. Since the recorded interferogram shows distortions caused by nonlinear response of the MCT-detector, especially, when a photoconductive detector is used, we implemented a software nonlinearity correction method proposed by Keens and Simon (1). The effects of the nonlinearity correction on the time-resolved step-scan difference spectra of biological samples are demonstrated.

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.

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.