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Biophys J, July 2001, p. 394-406, Vol. 81, No. 1

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

Christian Hackmann,* Jarmila Guijarro,* Igor Chizhov,dagger Martin Engelhard,dagger Christoph Rödig,* and Friedrich Siebert*

 *Sektion Biophysik, Institut für Molekulare Medizin und Zellforschung, Albert-Ludwigs-Universität, D-79104 Freiburg; and  dagger Max-Planck-Institut für Molekulare Physiologie, D-44227 Dortmund, Germany

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, L1, L2, and O. Between L1 and L2, 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 L2, we are unable to identify an N-like state. At very high chloride concentrations (>5 M), we observe a branching of the photocycle from L2 directly back to the dark state, and we provide evidence for direct back-isomerization from L2. This branching leads to the reported reduction of transport activity at such high chloride concentrations. We interpret the L1 to L2 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.

Biophys J, July 2001, p. 394-406, Vol. 81, No. 1
© 2001 by the Biophysical Society   0006-3495/01/07/394/13  $2.00


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