Time-resolved step-scan Fourier transform infrared spectroscopy reveals differences between early and late M intermediates of bacteriorhodopsin

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 similar to 3 mu s 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 similar to 40 mu s, 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 similar to 130 mu s), 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 VM equilibrium toward M and possibly to regulate the change of the accessibility of the Schiff base necessary for effective proton pumping.