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

2001; American Chemical Society; Volume: 40; Issue: 38 Linguagem: Inglês

10.1021/bi002651d

1943-295X

Jasper J. van Thor, Berthold Borucki, Wim Crielaard, Harald Otto, Tilman Lamparter, Jon Hughes, Klaas J. Hellingwerf, Maarten P. Heyn,

Light effects on plants

The Pr to Pfr 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 Pfr formed at pH 9 to between 0.6 (Cph1) and 1.1 (Cph1Δ2) H+/Pfr 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 Pfr 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 Pfr. The acidification observed in the Pr to Pfr 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 Pr/Pfr 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 Pr and Pfr with an approximate pKa of 9.5. The protonation state of the chromophore at neutral pH is therefore the same in both Pr and Pfr. The light-induced deprotonation and reprotonation of Cph1 at neutral pH are thus due to pKa 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.