Light-Induced Proton Release and Proton Uptake Reactions in the Cyanobacterial Phytochrome Cph1
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- 作者：Jasper J. van Thor ; Berthold Borucki ; Wim Crielaard ; Harald Otto ; Tilman Lamparter ; Jon Hughes ; Klaas J. Hellingwerf ; and Maarten P. Heyn
- 刊名：Biochemistry
- 出版年：2001
- 出版时间：September 25, 2001
- 年：2001
- 卷：40
- 期：38
- 页码：11460 - 11471
- 全文大小：145K
- 年卷期：v.40,no.38(September 25, 2001)
- ISSN：1520-4995
文摘
The Pr to Pfr transition of recombinant Synechocystis PCC 6803 phytochrome Cph1 and itsN-terminal sensor domain Cph1
2 is accompanied by net acidification in unbuffered solution. The extentof this net photoreversible proton release was measured with a conventional pH electrode and increasedfrom less than 0.1 proton released per Pfr formed at pH 9 to between 0.6 (Cph1) and 1.1 (Cph12) H+/Pfrat pH 6. The kinetics of the proton release were monitored at pH 7 and pH 8 using flash-induced transientabsorption measurements with the pH indicator dye fluorescein. Proton release occurs with time constantsof ~4 and ~20 ms that were also observed in parallel measurements of the photocycle (
3 and 4). Thenumber of transiently released protons per Pfr formed is about one. This H+ release phase is followed bya proton uptake phase of a smaller amplitude that has a time constant of ~270 ms (5) and is synchronouswith the formation of Pfr. The acidification observed in the Pr to Pfr transition with pH electrodes is thenet effect of these two sequential protonation changes. Flash-induced transient absorption measurementswere carried out with Cph1 and Cph12 at pH 7 and pH 8. Global analysis indicated the presence of fivekinetic components (1-5: 5 and 300 
s and 3, 30, and 300 ms). Whereas the time constants wereapproximately pH independent, the corresponding amplitude spectra (B1, B3, and B5) showed significantpH dependence. Measurements of the Pr/Pfr photoequilibrium indicated that it is pH independent in therange of 6.5-9.0. Analysis of the pH dependence of the absorption spectra from 6.5 to 9.0 suggested thatthe phycocyanobilin chromophore deprotonates at alkaline pH in both Pr and Pfr with an approximate pKaof 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 inthe protein moiety, which are linked to conformational transitions occurring around 4 and 270 ms afterphotoexcitation. These transient structural changes may be relevant for signal transduction by thiscyanobacterial phytochrome.
2 is accompanied by net acidification in unbuffered solution. The extentof this net photoreversible proton release was measured with a conventional pH electrode and increasedfrom less than 0.1 proton released per Pfr formed at pH 9 to between 0.6 (Cph1) and 1.1 (Cph12) H+/Pfrat pH 6. The kinetics of the proton release were monitored at pH 7 and pH 8 using flash-induced transientabsorption measurements with the pH indicator dye fluorescein. Proton release occurs with time constantsof ~4 and ~20 ms that were also observed in parallel measurements of the photocycle (3 and 4). Thenumber of transiently released protons per Pfr formed is about one. This H+ release phase is followed bya proton uptake phase of a smaller amplitude that has a time constant of ~270 ms (5) and is synchronouswith the formation of Pfr. The acidification observed in the Pr to Pfr transition with pH electrodes is thenet effect of these two sequential protonation changes. Flash-induced transient absorption measurementswere carried out with Cph1 and Cph12 at pH 7 and pH 8. Global analysis indicated the presence of fivekinetic components (1-5: 5 and 300 s and 3, 30, and 300 ms). Whereas the time constants wereapproximately pH independent, the corresponding amplitude spectra (B1, B3, and B5) showed significantpH dependence. Measurements of the Pr/Pfr photoequilibrium indicated that it is pH independent in therange of 6.5-9.0. Analysis of the pH dependence of the absorption spectra from 6.5 to 9.0 suggested thatthe phycocyanobilin chromophore deprotonates at alkaline pH in both Pr and Pfr with an approximate pKaof 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 inthe protein moiety, which are linked to conformational transitions occurring around 4 and 270 ms afterphotoexcitation. These transient structural changes may be relevant for signal transduction by thiscyanobacterial phytochrome.