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Biophys J, September 2001, p. 1600-1612, Vol. 81, No. 3

Temperature and Halide Dependence of the Photocycle of Halorhodopsin from Natronobacterium pharaonis

Igor Chizhov and Martin Engelhard

Max-Planck-Institut für Molekulare Physiologie, 44227 Dortmund, Germany

The photocycle kinetics of halorhodopsin from Natronobacterium pharaonis (pHR575) was analyzed at different temperatures and chloride concentrations as well as various halides. Over the whole range of modified parameters the kinetics can be adequately modeled with six apparent rate constants. Assuming a model in which the observed rates are assigned to irreversible transitions of a single relaxation chain, six kinetically distinguishable states (P1-6) are discernible that are formed from four chromophore states (spectral archetypes Sj: K570, L(N)520, O600, pHR'575). Whereas P1 coincides with K570 (S1), both P2 and P3 have identical spectra resembling L520 (S2), thus representing a true spectral silent transition between them. P4 constitutes a fast temperature-dependent equilibrium between the chromophore states S2 and S3 (L520 and O600, respectively). The subsequent equilibrium (P5) of the same spectral archetypes is only moderately temperature dependent but shows sensitivity toward the type of anion and the chloride concentration. Therefore, S2 and S3 occurring in P4 as well as in P5 have to be distinguished and are assigned to L520left-right-arrow O<UP><SUB>600</SUB><SUP>1</SUP></UP> and O<UP><SUB>600</SUB><SUP>2</SUP></UP>left-right-arrow N520 equilibrium, respectively. It is proposed that P4 and P5 represent the anion release and uptake steps. Based on the experimental data affinities of the halide binding sites are estimated.

Biophys J, September 2001, p. 1600-1612, Vol. 81, No. 3
© 2001 by the Biophysical Society   0006-3495/01/09/1600/13  $2.00


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