Static and time-resolved step-scan Fourier transform infrared investigations of the photoreaction of halorhodopsin from Natronobacterium pharaonis:: Consequences for models of the anion translocation mechanism

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.