The P r 3 P fr phototransformation of the bacteriophytochrome Agp1 from Agrobacterium tumefaciens and the structures of the biliverdin chromophore in the parent states and the cryogenically trapped intermediate Meta-R C were investigated with resonance Raman spectroscopy and flash photolysis. Strong similarities with the resonance Raman spectra of plant phytochrome A indicate that in Agp1 the methine bridge isomerization state of the chromophore is ZZZasa in P r and ZZEssa in P fr , with all pyrrole nitrogens being protonated. Photoexcitation of P r is followed by (at least) three thermal relaxation components in the formation of P fr with time constants of 230 s and 3.1 and 260 ms. H 2 O/D 2 O exchange reveals kinetic isotope effects of 1.9, 2.6, and 1.3 for the respective transitions that are accompanied by changes of the amplitudes. The second and the third relaxation correspond to the formation and decay of Meta-R C , respectively. Resonance Raman measurements of Meta-R C indicate that the chromophore adopts a deprotonated ZZE configuration. Measurements with a pH indicator dye show that formation and decay of Meta-R C are associated with proton release and uptake, respectively. The stoichiometry of the proton release corresponds to one proton per photoconverted molecule. The coupling of transient chromophore deprotonation and proton release, which is likely to be an essential element in the P r 3 P fr photoconversion mechanism of phytochromes in general, may play a crucial role for the structural changes in the final step of the P fr formation that switch between the active and the inactive state of the photoreceptor.Phytochromes are photoreceptors that utilize light as a source of information for controlling numerous biological processes (1, 2). The chromophore, a methine-bridged tetrapyrrole ( Fig. 1), acts as a photoswitch between two stable, spectrally distinct forms, denoted as P r and P fr according to the red and far-red absorption maxima, respectively. The P r /P fr interconversion is initiated by the rapid Z/E photoisomerization of the C-D methine bridge (3), followed by chromophore relaxations that are coupled to structural changes of the apoprotein (4). These structural changes are the trigger for signal transduction. Resonance Raman (RR) 2 and IR spectroscopy have provided valuable insight into light-induced chromophore and protein structural changes (e.g. see Refs. 5-10), but molecular and mechanistic details are not yet known and no crystal structure of a phytochrome has been reported so far.While phytochromes were originally thought to be restricted to plants, the discovery of these chromoproteins in cyanobacteria (11) and other bacteria points to the prokaryotic origin of this family of photoreceptors. In contrast to plant phytochromes, typical bacterial phytochromes are light-regulated histidine kinases. Despite the quite different regulatory functions (12, 13), plant and bacterial phytochromes exhibit structural and mechanistic similarities. The phytochromobilin chromophore of plant phytoc...

The mutants H250A and D197A of Agp1 phytochrome from Agrobacterium tumefaciens were prepared and investigated by different spectroscopic and biochemical methods. Asp-197 and His-250 are highly conserved amino acids and are part of the hydrogen-bonding network that involves the chromophore. Both substitutions cause a destabilization of the protonated chromophore in the Pr state as revealed by resonance Raman and UV-visible absorption spectroscopy. Titration experiments demonstrate a lowering of the pK a from 11.1 (wild type) to 8.8 in H250A and 7.2 in D197A. Photoconversion of the mutants does not lead to the Pfr state. H250A is arrested in a meta-Rc-like state in which the chromophore is deprotonated. For H250A and the wild-type protein, deprotonation of the chromophore in meta-Rc is coupled to the release of a proton to the external medium, whereas the subsequent proton re-uptake, linked to the formation of the Pfr state in the wild-type protein, is not observed for H250A. No transient proton exchange with the external medium occurs in D197A, suggesting that Asp-197 may be the proton release group. Both mutants do not undergo the photoinduced protein structural changes that in the wild-type protein are detectable by size exclusion chromatography. These conformational changes are, therefore, attributed to the meta-Rc 3 Pfr transition and most likely coupled to the transient proton re-uptake. The present results demonstrate that Asp-197 and His-250 are essential for stabilizing the protonated chromophore structure in the parent Pr state, which is required for the primary photochemical process, and for the complete photo-induced conversion to the Pfr state.

The P(r) to P(fr) transition of recombinant Synechocystis PCC 6803 phytochrome Cph1 and its N-terminal sensor domain Cph1Delta2 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 (Cph1Delta2) 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 approximately 4 and approximately 20 ms that were also observed in parallel measurements of the photocycle (tau(3) and tau(4)). 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 approximately 270 ms (tau(5)) 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 Cph1Delta2 at pH 7 and pH 8. Global analysis indicated the presence of five kinetic components (tau(1)-tau(5): 5 and 300 micros 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)) 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.

We studied the kinetics of proton uptake and release by photoactive yellow protein (PYP) from Ectothiorhodospira halophila in wild type and the E46Q and E46A mutants by transient absorption spectroscopy with the pH-indicator dyes bromocresol purple or cresol red in unbuffered solution. In parallel, we investigated the kinetics of chromophore protonation as monitored by the rise and decay of the blue-shifted state I(2) (lambda(max) = 355 nm). For wild type the proton uptake kinetics is synchronized with the fast phase of I(2) formation (tau = 500 micros at pH 6.2). The transient absorption signal from the dye also contains a slower component which is not due to dye deprotonation but is caused by dye binding to a hydrophobic patch that is transiently exposed in the structurally changed and partially unfolded I(2) intermediate. This conclusion is based on the wavelength, pH, and concentration dependence of the dye signal and on dye measurements in the presence of buffer. SVD analysis, moreover, indicates the presence of two components in the dye signal: protonation and dye binding. The dye binding has a rise time of about 4 ms and is coupled kinetically with a transition between two I(2) intermediates. In the mutant E46Q, which lacks the putative internal proton donor E46, the formation of I(2) is accelerated, but the proton uptake kinetics remains kinetically coupled to the fast phase of I(2) formation (tau = 100 micros at pH 6.3). For this mutant the protein conformational change, as monitored by the dye binding, occurs with about the same time constant as in wild type but with reduced amplitude. In the alkaline form of the mutant E46A the formation of the I(2)-like intermediate is even faster as is the proton uptake (tau = 20 micros at pH 8.3). No dye binding occurred in E46A, suggesting the absence of a conformational change. In all of the systems proton release is synchronized with the decay of I(2). Our results support mechanisms in which the chromophore of PYP is protonated directly from the external medium rather than by the internal donor E46.