Anion uptake in halorhodopsin from Natromonas pharaonis studied by FTIR spectroscopy: consequences for the anion transport mechanism.
The uptake of chloride, bromide, iodide, nitrate, and azide by anion-depleted blue halorhodopsin from Natronobacterium pharaonis has been followed by FTIR difference spectroscopy using an ATR sampling device. The spectra are compared with the spectrum of the O intermediate obtained by time-resolved FTIR studies of the photocycle. It is demonstrated that anion-free blue halorhodopsin can be identified with the O intermediate and, thus, that the decay of O is due to the passive uptake of the anion. The great similarity of the anion-binding spectra and their identity in the case of the monoatomic anions indicate a rather unspecific binding site for the different anions dominated by electrostatic interactions. Comparing spectra obtained with 15N nitrate and unlabeled nitrate, the NO-stretching bands could be identified. The small splitting and the small IR intensity of those bands indicate a rather nonpolar binding site with a rather isotropic influence on the nitrate, in contrast to aqueous nitrate. In further experiments on the photocycle of blue halorhodopsin, the all-trans --> 13-cis isomerization can be clearly identified. Up to 100 micros, the isomerization-induced structural changes deduced from amide I changes are similar to those occurring during the anion-transporting photocycle. Compared to these, the molecular changes involved in the release and their reversion during the uptake of anions are considerably larger. They can be reached via two pathways: (1) by reducing the anion concentration and (2) transiently during the anion-transporting photocycle with the formation of the precursor of O with O conformation. Consequences of the anion transport mechanism are discussed.
Guijarro
Jarmila
J
Sektion Biophysik, Institut für Molekulare Medizin und Zellforschung, Albert-Ludwigs-Universität, 79104 Freiburg, Germany.
Engelhard
Martin
M
Siebert
Friedrich
F
D001386:Azides / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism*; D025602:Halorhodopsins / Q000378:metabolism*; D017136:Ion Transport / Q000502:physiology*; D008027:Light; D019633:Natronobacterium / Q000378:metabolism*; D009566:Nitrates / Q000378:metabolism*; D011485:Protein Binding; D017550:Spectroscopy, Fourier Transform Infrared; D013997:Time Factors
D001386:Azides; D001426:Bacterial Proteins; D002712:Chlorides; D025602:Halorhodopsins; D009566:Nitrates