Controlled reduction of the humidity induces a shortcut recovery reaction in the photocycle of photoactive yellow protein

The photocycle of the blue-light photoreceptor protein Photoactive Yellow Protein (PYP) was studied at reduced relative humidity (RH). Photocycle kinetics and spectra were measured in thin films of PYP in which the relative humidity was set at values between 29 and 98% RH with saturated solutions of various salts. We show that in this range, approximately 200 water molecules per PYP molecule are released from the film. As humidity decreased, photocycle transition rates changed, until at low humidity (RH < 50%) an authentic photocycle was no longer observed and the absorption spectrum of the dark, equilibrium state of PYP started to shift to 355 nm, that is, to a form resembling that of pB(dark). At moderately reduced humidity (i.e., >50% RH), an authentic photocycle is still observed, although its characteristics differ from those in solution. As humidity decreases, the rate of ground state recovery increases, while the rate of depletion of the first red-shifted intermediate pR dramatically decreases. The latter observation contrasts all so-far known modulations of the rate of the transition of the red-shifted- to the blue-shifted intermediates of PYP, which is consistently accelerated by all other modulations of the mesoscopic context of the protein. Under these same conditions, the long-lived, blue-shifted intermediate was formed not only with slower kinetics than in solution but also to a smaller extent. Global analysis of these data indicates that in this low humidity environment the photocycle can take a different route than in solution, that is, part of pG recovers directly from pR. These experiments on wild-type PYP, in combination with observations on a variant of PYP obtained by site-directed mutagenesis (the E46Q mutant protein), further document the context dependence of the photocycle transitions of PYP and are relevant for the interpretation of results obtained in both spectroscopic and diffraction studies with crystalline PYP.     

Influence of the crystalline state on photoinduced dynamics of photoactive yellow protein studied by ultraviolet-visible transient absorption spectroscopy

Time-resolved ultraviolet-visible spectroscopy was used to characterize the photocycle transitions in single crystals of wild-type and the E-46Q mutant of photoactive yellow protein (PYP) with microsecond time resolution. The results were compared with the results of similar measurements on aqueous solutions of these two variants of PYP, with and without the components present in the mother liquor of crystals. The experimental data were analyzed with global and target analysis. Distinct differences in the reaction path of a PYP molecule are observed between these conditions when it progresses through its photocycle. In the crystalline state i), much faster relaxation of the late blue-shifted photocycle intermediate back to the ground state is observed; ii), this intermediate in crystalline PYP absorbs at 380 nm, rather than at 350-360 nm in solution; and iii), for various intermediates of this photocycle the forward reaction through the photocycle directly competes with a branching reaction that leads directly to the ground state. Significantly, with these altered characteristics, the spectroscopic data on PYP are fully consistent with the structural data obtained for this photoreceptor protein with time-resolved x-ray diffraction analysis, particularly for wild-type PYP.     

The two photocycles of photoactive yellow protein from Rhodobacter sphaeroides

The absorption spectrum of the photoactive yellow protein from Rhodobacter sphaeroides (R-PYP) shows two maxima, absorbing at 360 nm (R-PYP(360)) and 446 nm (R-PYP(446)), respectively. Both forms are photoactive and part of a temperature- and pH-dependent equilibrium (Haker, A., Hendriks, J., Gensch, T., Hellingwerf, K. J., and Crielaard, W. (2000) FEBS Lett. 486, 52-56). At 20 degrees C, for PYP characteristic, the 446-nm absorbance band displays a photocycle, in which the depletion of the 446-nm ground state absorption occurs in at least three phases, with time constants of <30 ns, 0.5 micros, and 17 micros. Intermediates with both blue- and red-shifted absorption maxima are transiently formed, before a blue-shifted intermediate (pB(360), lambda(max) = 360 nm) is established. The photocycle is completed with a monophasic recovery of the ground state with a time constant of 2.5 ms. At 7 degrees C these photocycle transitions are slowed down 2- to 3-fold. Upon excitation of R-PYP(360) with a UV-flash (330 +/- 50 nm) a species with a difference absorption maximum at approximately 435 nm is observed that returns to R-PYP(360) on a minute time scale. Recovery can be accelerated by a blue light flash (450 nm). R-PYP(360) and R-PYP(446) differ in their overall protein conformation, as well as in the isomerization and protonation state of the chromophore, as determined with the fluorescent polarity probe Nile Red and Fourier Transform Infrared spectroscopy, respectively.     

Thermochromatium tepidum photoactive yellow protein/bacteriophytochrome/diguanylate cyclase: characterization of the PYP domain

The purple phototrophic bacterium, Thermochromatium tepidum, contains a gene for a chimeric photoactive yellow protein/bacteriophytochrome/diguanylate cyclase (Ppd). We produced the Tc. tepidum PYP domain (Tt PYP) in Escherichia coli, and found that it has a wavelength maximum at 358 nm due to a Leu46 substitution of the color-tuning Glu46 found in the prototypic Halorhodospira halophila PYP (Hh PYP). However, the 358 nm dark-adapted state is in a pH-dependent equilibrium with a yellow species absorbing at 465 nm (pK(a) = 10.2). Following illumination at 358 nm, photocycle kinetics are characterized at pH 7.0 by a small bleach and red shift to what appears to be a long-lived cis intermediate (comparable to the I(2) intermediate in Hh PYP). The recovery to the dark-adapted state has a lifetime of approximately 4 min, which is approximately 1500 times slower than that for Hh PYP. However, when the Tt PYP is illuminated at pH values above 7.5, the light-induced difference spectrum indicates a pH-dependent equilibrium between the I(2) intermediate and a red-shifted 440 nm intermediate. This equilibrium could be responsible for the sigmoidal pH dependence of the recovery of the dark-adapted state (pK(a) = 8.8). In addition, the light-induced difference spectrum shows that, at pH values above 9.3, there is an apparent bleach near 490 nm superimposed on the 358 and 440 nm changes, which we ascribe to the equilibrium between the protonated and ionized dark-adapted forms. The L46E mutant of Tt PYP has a wavelength maximum at 446 nm, resembling wild-type Hh PYP. The kinetics of recovery of L46E following illumination with white light are slow (lifetime of 15 min at pH 7), but are comparable to those of wild-type Tt PYP. We conclude that Tt PYP is unique among the PYPs studied to date in that it has a photocycle initiated from a dark-adapted state with a protonated chromophore at physiological pH. However, it is kinetically most similar to Rhodocista centenaria PYP (Ppr) despite the very different absorption spectra due to the lack of E46.     

Site-specific mutations provide new insights into the origin of pH effects and alternative spectral forms in the photoactive yellow protein from Halorhodospira halophila

Acid/base titrations of wild-type PYP and mutants, either in buffer or in the presence of chaotropes such as thiocyanate, establish the presence of four spectral forms including the following: a neutral form (446-476 nm), an acidic form (350-355 nm), an alkaline form (430-440 nm), and an intermediate wavelength form (355-400 nm). The acidic species is formed by protonation of the oxyanion of the para-hydroxy-cinnamyl cysteine chromophore as a secondary result of acid denaturation (with pK(a) values of 2.8-5.4) and often results in precipitation of the protein, and in the case of wild-type PYP, eventual hydrolysis of the chromophore thioester bond at pH values below 2. Thus, the large and complex structural changes associated with the acidic species make it a poor model for the long-lived photocycle intermediate, I(2), which undergoes more moderate structural changes. Mutations at E46, which is hydrogen-bonded to the chromophore, have only two spectral forms accessible to them, the neutral and the acidic forms. Thus, an intact E46 carboxyl group is essential for observation of either intermediate or alkaline wavelength forms. The alkaline form is likely to be due to ionization of E46 in the folded protein. We postulate that the intermediate wavelength form is due to a conformational change that allows solvent access to E46 and formation of a hydrogen-bond from a water molecule to the carboxylic acid group, thus weakening its interaction with the chromophore. Increasing solvent access to the intermediate spectral form with denaturant concentration results in a continuously blue-shifted wavelength maximum.     

Time-resolved X-ray diffraction study of structural changes associated with the photocycle of bacteriorhodopsin.

The time course of structural changes accompanying the transition from the M412 intermediate to the BR568 ground state in the photocycle of bacteriorhodopsin (BR) from Halobacterium halobium was studied at room temperature with a time resolution of 15 ms using synchrotron radiation X-ray diffraction. The M412 decay rate was slowed down by employing mutated BR Asp96Asn in purple membranes at two different pH-values. The observed light-induced intensity changes of in-plane X-ray reflections were fully reversible. For the mutated BR at neutral pH the kinetics of the structural alterations (tau 1/2 = 125 ms) were very similar to those of the optical changes characterizing the M412 decay, whereas at pH 9.6 the structural relaxation (tau 1/2 = 3 s) slightly lagged behind the absorbance changes at 410 nm. The overall X-ray intensity change between the M412 intermediate and the ground state was about 9% for the different samples investigated and is associated with electron density changes close to helix G, B and E. Similar changes (tau 1/2 = 1.3-3.6 s), which also confirm earlier neutron scattering results on the BR568 and M412 intermediates trapped at -180 degrees C, were observed with wild type BR retarded by 2 M guanidine hydrochloride (pH 9.4). The results unequivocally prove that the tertiary structure of BR changes during the photocycle.     

Kinetics of proton uptake and dye binding by photoactive yellow protein in wild type and in the E46Q and E46A mutants

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.     

Light-induced unfolding of photoactive yellow protein mutant M100L

Light-activation of the PAS domain protein photoactive yellow protein (PYP) is believed to trigger a negative phototactic response in the phototropic bacterium Halorhodospira halophila. To investigate transient conformational changes of the PYP photocycle, we utilized the PYP mutant M100L that displays an increased lifetime of the putative signaling-state photointermediate PYP(M) by 3 orders of magnitude, as previously reported for the M100A mutant [Devanathan, S., Genick, U. K., Canestrelli, I. L., Meyer, T. E., Cusanovich, M. A., Getzoff, E. D., and Tollin, G. Biochemistry (1998) 37, 11563-11568]. The FTIR difference spectrum of PYP(M) and the ground state of M100L demonstrated extensive peptide-backbone structural changes as observed in the FTIR difference spectrum of the wild-type protein and PYP(M). The conformational change investigated by CD spectroscopy in the far-UV region showed reduction of the alpha-helical content by approximately 40%, indicating a considerable amount of changes in the secondary structure. The optical activity of the p-coumaric acid chromophore completely vanished upon PYP(M) in contrast to the dark state, indicating deformation of the binding pocket structure in PYP(M). The tertiary structural changes were further monitored by small-angle X-ray scattering measurements, which demonstrated a significant increase of the radius of gyration of the molecule by approximately 5% in PYP(M). These structural changes were reversed concomitantly with the chromophore anionization upon the dark state recovery. The observed changes of the quantities provided a more vivid view of the structural changes of the mutant PYP in going from PYP(M) to PYP(dark), which can be regarded as a process of folding of the secondary and the tertiary structures of the "PAS" domain structure, coupled with the p-coumaric acid chromophore deprotonation and isomerization.     

Picosecond decay kinetics and quantum yield of fluorescence of the photoactive yellow protein from the halophilic purple phototrophic bacterium, Ectothiorhodospira halophila

The photoactive yellow protein (PYP) has been previously shown to be partially bleached and red shifted (in less than 10 ns) by a pulse of laser excitation at the wavelength maximum (445 nm), to further bleach (k = 7.5 × 10³ s^-1), and then to slowly recover in the dark (k = 2.6 s^-1) (Meyer, T. E., G. Tollin, J. H. Hazzard, and M. A. Cusanovich. 1989. Biophys. J. 56:559-564). The quantum yield for the formation of the fully bleached form was found to be 0.64. We have now shown that the yellow protein is weakly fluorescent with an emission maximum at 495 nm (which mirrors excitation at 445 nm) and a fluorescence quantum yield of 1.4 × 10^-3. Measurement of the picosecond kinetics of the fluorescence decay shows that ~90% of the emission occurs with a lifetime of 12 ps. This is in good agreement with the quantum yield determination, which suggests that a single quenching process (presumably the photochemical event) is primarily responsible for the excited state decay. The lifetime of the excited state of PYP is remarkably similar to that for the rise of the first photochemical intermediate of bacteriorhodopsin, and underscores the fundamental similarity in their photocycles despite a lack of structural relationship.     

Resonance Raman spectroscopy and quantum chemical calculations reveal structural changes in the active site of photoactive yellow protein

Photoactive yellow protein (PYP) is a bacterial photoreceptor containing a 4-hydroxycinnamyl chromophore. Photoexcitation of PYP triggers a photocycle that involves at least two intermediate states: an early red-shifted PYP(L) intermediate and a long-lived blue-shifted PYP(M) intermediate. In this study, we have explored the active site structures of these intermediates by resonance Raman spectroscopy. Quantum chemical calculations based on a density functional theory are also performed to simulate the observed spectra. The obtained structure of the chromophore in PYP(L) has cis configuration and no hydrogen bond at the carbonyl oxygen. In PYP(M), the cis chromophore is protonated at the phenolic oxygen and forms the hydrogen bond at the carbonyl group. These results allow us to propose structural changes of the chromophore during the photocycle of PYP. The chromophore photoisomerizes from trans to cis configuration by flipping the carbonyl group to form PYP(L) with minimal perturbation of the tightly packed protein interior. Subsequent conversion to PYP(M) involves protonation on the phenolic oxygen, followed by rotation of the chromophore as a whole. This large motion of the chromophore is potentially correlated with the succeeding global conformational changes in the protein, which ultimately leads to transduction of a biological signal.     

New photocycle intermediates in the photoactive yellow protein from Ectothiorhodospira halophila: picosecond transient absorption spectroscopy.

Previous studies have shown that the room temperature photocycle of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila involves at least two intermediate species: I1, which forms in <10 ns and decays with a 200-micros lifetime to I2, which itself subsequently returns to the ground state with a 140-ms time constant at pH 7 (Genick et al. 1997. Biochemistry. 36:8-14). Picosecond transient absorption spectroscopy has been used here to reveal a photophysical relaxation process (stimulated emission) and photochemical intermediates in the PYP photocycle that have not been reported previously. The first new intermediate (I0) exhibits maximum absorption at approximately 510 nm and appears in </=3 ps after 452 nm excitation (5 ps pulse width) of PYP. Kinetic analysis shows that I0 decays with a 220 +/- 20 ps lifetime, forming another intermediate (Idouble dagger0) that has a similar difference wavelength maximum, but with lower absorptivity. Idouble dagger0 decays with a 3 +/- 0.15 ns time constant to form I1. Stimulated emission from an excited electronic state of PYP is observed both within the 4-6-ps cross-correlation times used in this work, and with a 16-ps delay for all probe wavelengths throughout the 426-525-nm region studied. These transient absorption and emission data provide a more detailed understanding of the mechanistic dynamics occurring during the PYP photocycle.     

Kinetics of and intermediates in a photocycle branching reaction of the photoactive yellow protein from Ectothiorhodospira halophila.

We have studied the kinetics of the blue light-induced branching reaction in the photocycle of photoactive yellow protein (PYP) from Ectothiorhodospira halophila, by nanosecond time-resolved UV/Vis spectroscopy. As compared to the parallel dark recovery reaction of the presumed blue-shifted signaling state pB, the light-induced branching reaction showed a 1000-fold higher rate. In addition, a new intermediate was detected in this branching pathway, which, compared to pB, showed a larger extinction coefficient and a blue-shifted absorption maximum. This substantiates the conclusion that isomerization of the chromophore is the rate-controlling step in the thermal photocycle reactions of PYP and implies that absorption of a blue photon leads to cis-->trans isomerization of the 4-hydroxy-cinnamyl chromophore of PYP in its pB state.     

Dual photoactive species in Glu46Asp and Glu46Ala mutants of photoactive yellow protein: a pH-driven color transition.

Photoactive yellow protein (PYP) is a blue light sensor present in the purple photosynthetic bacterium Ectothiorhodospira halophila, which undergoes a cyclic series of absorbance changes upon illumination at its lambda(max) of 446 nm. The anionic p-hydroxycinnamoyl chromophore of PYP is covalently bound as a thiol ester to Cys69, buried in a hydrophobic pocket, and hydrogen-bonded via its phenolate oxygen to Glu46 and Tyr42. The chromophore becomes protonated in the photobleached state (I(2)) after it undergoes trans-cis isomerization, which results in breaking of the H-bond between Glu46 and the chromophore and partial exposure of the phenolic ring to the solvent. In previous mutagenesis studies of a Glu46Gln mutant, we have shown that a key factor in controlling the color and photocycle kinetics of PYP is this H-bonding system. To further investigate this, we have now characterized Glu46Asp and Glu46Ala mutants. The ground-state absorption spectrum of the Glu46Asp mutant shows a pH-dependent equilibrium (pK = 8.6) between two species: a protonated (acidic) form (lambda(max) = 345 nm), and a slightly blue-shifted deprotonated (basic) form (lambda(max) = 444 nm). Both of these species are photoactive. A similar transition was also observed for the Glu46Ala mutant (pK = 7.9), resulting in two photoactive red-shifted forms: a basic species (lambda(max) = 465 nm) and a protonated species (lambda(max) = 365 nm). We attribute these spectral transitions to protonation/deprotonation of the phenolate oxygen of the chromophore. This is demonstrated by FT Raman spectra. Dark recovery kinetics (return to the unphotolyzed state) were found to vary appreciably between these various photoactive species. These spectral and kinetic properties indicate that the hydrogen bond between Glu46 and the chromophore hydroxyl group is a dominant factor in controlling the pK values of the chromophore and the glutamate carboxyl.     

On the involvement of single-bond rotation in the primary photochemistry of photoactive yellow protein

Prior experimental observations, as well as theoretical considerations, have led to the proposal that C₄-C₇ single-bond rotation may play an important role in the primary photochemistry of photoactive yellow protein (PYP). We therefore synthesized an analog of this protein's 4-hydroxy-cinnamic acid chromophore, (5-hydroxy indan-(1E)-ylidene)acetic acid, in which rotation across the C₄-C₇ single bond has been locked with an ethane bridge, and we reconstituted the apo form of the wild-type protein and its R52A derivative with this chromophore analog. In PYP reconstituted with the rotation-locked chromophore, 1), absorption spectra of ground and intermediate states are slightly blue-shifted; 2), the quantum yield of photochemistry is ∼60% reduced; 3), the excited-state dynamics of the chromophore are accelerated; and 4), dynamics of the thermal recovery reaction of the protein are accelerated. A significant finding was that the yield of the transient ground-state intermediate in the early phase of the photocycle was considerably higher in the rotation-locked samples than in the corresponding samples reconstituted with p-coumaric acid. In contrast to theoretical predictions, the initial photocycle dynamics of PYP were observed to be not affected by the charge of the amino acid residue at position 52, which was varied by 1), varying the pH of the sample between 5 and 10; and 2), site-directed mutagenesis to construct R52A. These results imply that C₄-C₇ single-bond rotation in PYP is not an alternative to C₇=C₈ double-bond rotation, in case the nearby positive charge of R52 is absent, but rather facilitates, presumably with a compensatory movement, the physiological Z/E isomerization of the blue-light-absorbing chromophore.     

Photoreversal kinetics of the I1 and I2 intermediates in the photocycle of photoactive yellow protein by double flash experiments with variable time delay

We investigated the kinetics of photoreversal from the I(1) and I(2) intermediates of photoactive yellow protein (PYP) by time-resolved optical absorption spectroscopy with double flash excitation. A first flash, at 430 nm, initiated the photocycle. After a variable time delay, the I(1) intermediate was photoreversed by a second flash, at 500 nm, or a mixture of I(2) and I(2)' intermediates was photoreversed by a second flash, at 355 nm. By varying the delay from 1 micros to 3 s, we were able to selectively excite the intermediates I(1), I(2), and I(2)'. The photoreversal kinetics of I(2) and I(2)' at 21 different delays and two wavelengths (340 and 450 nm) required two exponentials for a global fit with time constants of tau(1) = 57 +/- 5 micros and tau(2) = 380 +/- 40 micros (pH 6, 20 degrees C). These were assigned to photoreversal from sequential I(2) and I(2)' intermediates, respectively. The good agreement of the delay dependence of the two amplitudes, A(1) and A(2), with the time dependence of the I(2) and I(2)' populations provided strong evidence for the sequential model. The persistence of A(1) beyond delay times of 5 ms and its decay, together with A(2) around 500 ms, suggest moreover that I(2) and I(2)' are in thermal equilibrium. The wavelength dependence of the photoreversal kinetics was measured at 26 wavelengths from 510 to 330 nm at the two fixed delays of 1 and 10 ms. These data also required two exponentials for a global fit with tau(1) = 59 +/- 5 micros and tau(2) = 400 +/- 40 micros, in good agreement with the delay results. Photoreversal from I(2)' is slower than from I(2), since, in addition to chromophore protonation, the global conformational change has to be reversed. Our data thus provide a first estimate of about 59 micros for deprotonation and 400 micros for the structural change, which also occurs in the thermal decay of the signaling state but is obscured there since reisomerization is rate-limiting. The first step in photoreversal is rapid cis-trans isomerization of the chromophore, which we could not resolve, but which was detected by the instantaneous increase in absorbance between 330 and 380 nm. In agreement with this observation, the spectrum of the I(2)'(trans) intermediate, derived from the A(2) amplitude spectrum, has a much larger extinction coefficient than the spectrum of the I(2)'(cis) intermediate. With a first flash, at 430 nm, and a second flash, at 500 nm, we observed efficient photoreversal of the I(1) intermediate at a delay of 20 micros when most molecules in the cycle are in I(1). We conclude that each of the three intermediates studied can be reversed by a laser flash. Depending on the progression of the photocycle, reversal becomes slower with the time delay, thus mirroring the individual steps of the forward photocycle.     

Amino acids in the N-terminal region regulate the photocycle of photoactive yellow protein.

The spectroscopic properties of photoactive yellow protein (PYP) partially digested by chymotrypsin were studied. Chymotrypsin yielded three major products that were yellow but distinguishable by SDS-PAGE. They were readily separated by DEAE-Sepharose column chromatography. Protein sequencing and mass spectrometry demonstrated that chymotrypsin cleaved the N-terminal 6, 15, or 23 amino acids (T6, T15, and T23). The blue-shifts of the absorption maxima and the increases in the apparent pK(a) of the chromophores relative to those of intact PYP were less than 4 nm and 0.2, respectively. The absorption spectra of the near-UV intermediates produced from T6, T15, and T23 were identical to that of intact PYP, but with lifetimes that were 140, 2,300, and 4,500 times longer, respectively. These observations suggest that the recovery of the dark state of PYP from the near-UV intermediate is accelerated by the N-terminal region, and that this region acts as a regulatory factor for the photocycle of PYP.     

Protonation/deprotonation reactions triggered by photoactivation of photoactive yellow protein from Ectothiorhodospira halophila.

Light-dependent pH changes were measured in unbuffered solutions of wild type photoactive yellow protein (PYP) and its H108F and E46Q variants, using two independent techniques: transient absorption changes of added pH indicator dyes and direct readings with a combination pH electrode. Depending on the absolute pH of the sample, a reversible protonation as well as a deprotonation can be observed upon formation of the transient, blue-shifted photocycle intermediate (pB) of this photoreceptor protein. The latter is observed at very alkaline pH, the former at acidic pH values. At neutral pH, however, the formation of the pB state is not paralleled by significant protonation/deprotonation of PYP, as expected for concomitant protonation of the chromophore and deprotonation of Glu-46 during pB formation. We interpret these results as further evidence that a proton is transferred from Glu-46 to the coumaric acid chromophore of PYP, during pB formation. One cannot exclude the possibility, however, that this transfer proceeds through the bulk aqueous phase. Simultaneously, an amino acid side chain(s) (e.g. His-108) changes from a buried to an exposed position. These results, therefore, further support the idea that PYP significantly unfolds in the pB state and resolve the controversy regarding proton transfer during the PYP photocycle.     

Photochemistry of two rhodopsinlike pigments in bacteriorhodopsin-free mutant of Halobacterium halobium.

Two photocycles due to two different pigments were found in membrane vesicles of a bacteriorhodopsin-free mutant of Halobacterium halobium. A pigment absorbing approximately 590 nm halorhodopsin (HR) underwent a faster photocycle with a phototransient at approximately 490 nm (half-time of decay, tau 1/2 = 10 ms). Another third rhodopsinlike pigment (TR) absorbing approximately 580 nm underwent a slower photocycle accompanying a phototransient absorbing below 410 nm (tau 1/2 = 0.8s). The photocycles were measured under various conditions of temperature, NaCl concentration, pH, and in the presence of cholate. All results obtained support the notion that the two photocycles are independent of each other, and the fast or the slow cycle can be abolished after these treatments. At alkaline pH, the wavelength of maximum absorbance of both pigments shifted to blue, but the magnitude of the shift of the pigment undergoing the slow photocycle was much greater than the other. The ratio of the content of the two pigments varies among bacteriorhodopsin-free mutants.     

Primary photoreaction of photoactive yellow protein studied by subpicosecond-nanosecond spectroscopy

The primary photochemical event of photoactive yellow protein (PYP) was studied by laser flash photolysis experiments on a subpicosecond-nanosecond time scale. PYP was excited by a 390-nm pulse, and the transient difference absorption spectra were recorded by a multichannel spectrometer for a more reliable spectral analysis than previously possible. Just after excitation, an absorbance decrease due to the stimulated emission at 500 nm and photoconversion of PYP at 450 nm were observed. The stimulated emission gradually shifted to 520 nm and was retained up to 4 ps. Then, the formation of a red-shifted intermediate with a broad absorption spectrum was observed from 20 ps to 1 ns. Another red-shifted intermediate with a narrow absorption spectrum was formed after 2 ns and was stable for at least 5 ns. The latter is therefore believed to correspond to I1 (PYP(L)), which has been detected on a nanosecond time scale or trapped at -80 degrees C. Singular value decomposition analysis demonstrated that the spectral shifts observed from 0.5 ps to 5 ns could be explained by two-component decay of excited state(s) and conversion from PYP(B) to PYP(L). The amount of PYP(L) at 5 ns was less than that of photoconverted PYP, suggesting the formation of another intermediate, PYP(H). In addition, the absorption spectra of these intermediates were calculated based on the proposed reaction scheme. Together, these results indicate that the photocycle of PYP at room temperature has a branched pathway in the early stage and is essentially similar to that observed under low-temperature spectroscopy.     

Early intermediates in the photocycle of the Glu46Gln mutant of photoactive yellow protein: femtosecond spectroscopy

Transient absorption spectroscopy in the time range from -1 ps to 4 ns, and over the wavelength range from 420 to 550 nm, was applied to the Glu46Gln mutant of the photoactive yellow protein (PYP) from Ectothiorhodospira halophila. This has allowed us to elucidate the kinetic constants of excited state formation and decay and photochemical product formation, and the spectral characteristics of stimulated emission and the early photocycle intermediates. Both the quantum efficiency ( approximately 0.5) and the rate constants for excited state decay and the formation of the initial photochemical intermediate (I(0)) were found to be quite similar to those obtained for wild-type PYP. In contrast, the rate constants for the formation of the subsequent photocycle intermediates (I(0)(double dagger) and I(1)), as well as for I(2) and for ground state regeneration as determined in earlier studies, were found to be from 3- to 30-fold larger. The structural implications of these results are discussed.