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The Nitrate Transporting Photochemical Reaction Cycle of the Pharaonis Halorhodopsin

Zoltán Bálint ^*, Melinda Lakatos ^*, Constanta Ganea , Janos K. Lanyi  and György Váró ^*

^* Institute of Biophysics, Biological Research Center of the Hungarian Academy of Sciences, Szeged, H-6701, Hungary; Department of Biophysics, University of Medicine "Carol Davila", 76241 Bucharest, Romania; and Department of Physiology and Biophysics, University of California, Irvine, California USA

Correspondence: Address reprint requests to György Váró, E-mail: varo{at}nucleus.szbk.u-szeged.hu.

Time-resolved spectroscopy, absorption kinetic and electric signal measurement techniques were used to study the nitrate transporting photocycle of the pharaonis halorhodopsin. The spectral titration reveals two nitrate-binding constants, assigned to two independent binding sites. The high-affinity binding site (K_a = 11 mM) contributes to the appearance of the nitrate transporting photocycle, whereas the low-affinity constant (having a K_a of 7 M) slows the last decay process in the photocycle. Although the spectra of the intermediates are not the same as those found in the chloride transporting photocycle, the sequence of the intermediates and the energy diagrams are similar. The differences in spectra and energy levels can be attributed to the difference in the size of the transported chloride or nitrate. Electric signal measurements show that a charge is transferred across the membrane during the photocycle, as expected. A new observation is an apparent release and rebinding of a small fraction of the retinal, inside the retinal pocket, during the photocycle. The release occurs during the N-to-O transition, whereas the rebinding happens in several seconds, well after the other steps of the photocycle are over.

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