Time-Resolved Step-Scan Fourier Transform Infrared Spectroscopy Reveals Differences between Early and Late M Intermediates of Bacteriorhodopsin

Abstract

In this report, from time-resolved step-scan Fourier transform infrared investigations from 15 ns to 160 ms, we provide evidence for the subsequent rise of three different M states that differ in their structures. The first state rises with approximately 3 microseconds 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 approximately 40 microseconds, has almost all of the characteristics of the "late" M state, i.e., it differs considerably from the first one. Here, the L left arrow over right arrow M equilibrium is shifted toward M, although some percentage of L still persists. In the last M state (rise time approximately 130 microseconds), 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.

Water Structural Changes in the L and M Photocycle Intermediates of Bacteriorhodopsin as Revealed by Time-Resolved Step-Scan Fourier Transform Infrared (FTIR) Spectroscopy

“…This is probably the sum of a b 5 -like spectrum with a spectrum that has a nearly mirror-image shape (restoration of the initial BR state from the last step of the photocycle). It is very similar to the final spectrum shown by Rödig et al (28). We plan to discuss this spectrum in a separate publication.…”

“…In this spectral region (1900 to 900 cm −1 ), all of the spectra from films hydrated with H 2 18 O (not shown) coincided with the spectra from films hydrated with H 2 16 O shown in Figure 2. The spectral shapes of a series of our b-spectra are also similar to those of Rödig et al (28). In figure 2(i), the residuals (average of absolute value of the difference between fit and data) are depicted as a pair of curves, showing the positive value of the residuals, and its negative, at each frequency.…”

“…Previously Rödig et al (28) have shown that the Schiff base HOOP band of L at room temperature is located at 983 cm −1 , while Maeda et al (27) have shown that the 1074, 1065 and 1057 cm −1 bands of L at 170 K are due to the HOOP bands of the Schiff base. Figure 5 shows an L-minus-BR spectrum (a) that was obtained by subtracting the M-minus-BR spectrum (20% of b 3 ) and the contribution of K L (50% of b 0 ) from b 1 as described above, together with the L-minus-BR spectrum at 170 K (b).…”

“…However, some of these interactions may be different at room temperature than at cryogenic temperatures where these observations were originally made. This possibility is suggested by the observation that the hydrogen out-of-plane bending vibration (HOOP) of the Schiff base in L at 170 K (27) is different from the ones detected in L at room temperature (28). Furthermore, the L intermediate produced by photoreaction at 170 K forms little or no M intermediate upon warming of the sample at least at neutral pH, and instead returns to the unphotolyzed BR state (29,30).…”

“…Until recently, time-resolved FTIR spectroscopic studies (9,28,(31)(32)(33)(34) focused on changes in the 1800-800 cm −1 frequency region, which includes information about protein residues and the chromophore during the photocycle, but did not deal with the vibration bands of water molecules. Gerwert and his colleagues have successfully obtained time-resolved FTIR difference spectra at room temperature for two broad vibrational features, one in the 2200-1700 cm −1 region (35,36) and the other in the 2900-2500 cm −1 region (35,37).…”