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Article

Light-Induced Proton Release and Proton Uptake Reactions in the Cyanobacterial Phytochrome Cph1^†

Jasper J. van Thor,*^‡^§ Berthold Borucki,^§ Wim Crielaard,^‡ Harald Otto, Tilman Lamparter, Jon Hughes,^# Klaas J. Hellingwerf,^‡ and Maarten P. Heyn

Laboratory for Microbiology, Swammerdam Institute for Life Sciences, University of Amsterdam, Nieuwe Achtergracht 166, 1018 WV Amsterdam, The Netherlands, Biophysics Group, Department of Physics, Freie Universität Berlin, Arnimallee 14, D-14195 Berlin, Germany, and Plant Physiology, Freie Universität Berlin, Königin-Luise-Strasse 12-16, D-14195 Berlin, Germany

Biochemistry, 2001, 40 (38), pp 11460–11471

DOI: 10.1021/bi002651d

Publication Date (Web): August 29, 2001

†

This work was supported by grants from the Deutsche Forschungsgemeinschaft to T.L. (Sfb 498, TP B2), J.H. (Sfb 498, TP B2), and M.P.H. (Sfb 498, TP B1).

To whom correspondence should be addressed. Present address: University of Oxford, Laboratory of Molecular Biophysics, Rex Richards Building, South Parks Road, Oxford OX1 3QU, U.K. Phone: +44-(0)1865-275374; fax: +44-(0)1865-275182; e-mail: jasper@biop.ox.ac.uk.

‡

University of Amsterdam.

These authors contributed equally to this work.

‖

Department of Physics, Freie Universität Berlin.

⊥

Plant Physiology, Freie Universität Berlin.

Present address: Plant Physiology, University of Giessen, D-35392 Giessen, Germany.

Abstract

The P_r to P_fr transition of recombinant Synechocystis PCC 6803 phytochrome Cph1 and its N-terminal sensor domain Cph1Δ2 is accompanied by net acidification in unbuffered solution. The extent of this net photoreversible proton release was measured with a conventional pH electrode and increased from less than 0.1 proton released per P_fr formed at pH 9 to between 0.6 (Cph1) and 1.1 (Cph1Δ2) H⁺/P_fr at pH 6. The kinetics of the proton release were monitored at pH 7 and pH 8 using flash-induced transient absorption measurements with the pH indicator dye fluorescein. Proton release occurs with time constants of 4 and 20 ms that were also observed in parallel measurements of the photocycle (τ₃ and τ₄). The number of transiently released protons per P_fr formed is about one. This H⁺ release phase is followed by a proton uptake phase of a smaller amplitude that has a time constant of 270 ms (τ₅) and is synchronous with the formation of P_fr. The acidification observed in the P_r to P_fr transition with pH electrodes is the net effect of these two sequential protonation changes. Flash-induced transient absorption measurements were carried out with Cph1 and Cph1Δ2 at pH 7 and pH 8. Global analysis indicated the presence of five kinetic components (τ₁−τ₅: 5 and 300 μs and 3, 30, and 300 ms). Whereas the time constants were approximately pH independent, the corresponding amplitude spectra (B₁, B₃, and B₅) showed significant pH dependence. Measurements of the P_r/P_fr photoequilibrium indicated that it is pH independent in the range of 6.5−9.0. Analysis of the pH dependence of the absorption spectra from 6.5 to 9.0 suggested that the phycocyanobilin chromophore deprotonates at alkaline pH in both P_r and P_fr with an approximate pK_a of 9.5. The protonation state of the chromophore at neutral pH is therefore the same in both P_r and P_fr. The light-induced deprotonation and reprotonation of Cph1 at neutral pH are thus due to pK_a changes in the protein moiety, which are linked to conformational transitions occurring around 4 and 270 ms after photoexcitation. These transient structural changes may be relevant for signal transduction by this cyanobacterial phytochrome.