Light-induced proton release and proton uptake reactions in the cyanobacterial phytochrome Cph1

The P-r to P-fr transition of recombinant Synechocystis PCC 6803 phytochrom e Cph1 and its N-terminal sensor domain Cph1 Delta2 is accompanied by net a cidification 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 (Cph1 Delta2) H+/P-fr at pH 6. The kinetics of the proton re lease were monitored at pH 7 and pH 8 using flash-induced transient absorpt ion measurements with the pH indicator dye fluorescein. Proton release occu rs with time constants of similar to4 and similar to 20 ms that were also o bserved in parallel measurements of the photocycle (tau (3) and tau (4)). T he number of transiently released protons per P-fr formed is about one. Thi s H+ release phase is followed by a proton uptake phase of a smaller amplit ude that has a time constant of similar to 270 ms (tau (5)) and is synchron ous 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 c arried out with Cph1 and Cph1 Delta2 at pH 7 and pH 8. Global analysis indi cated the presence of five kinetic components (tau (1)-tau (5): 5 and 300 m us and 3, 30, and 300 ms). Whereas the time constants were approximately pH independent, the corresponding amplitude spectra (B-1, B-3, and B-5) showe d 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 th e pH dependence of the absorption spectra from 6.5 to 9.0 suggested that th e 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 chromoph ore at neutral pH is therefore the same in both P-r and P-fr The light-indu ced deprotonation and reprotonation of Cph1 at neutral pH are thus due to p K(a) changes in the protein moiety, which are linked to conformational tran sitions occurring around 4 and 270 ms after photoexcitation. These transien t structural changes may be relevant for signal transduction by this cyanob acterial phytochrome.