pH Dependence of the photocycle kinetics of the E46Q mutant of photoactive yellow protein: protonation equilibrium between I1 and I2 intermediates,chromophore deprotonation by hydroxyl uptake,and protonation relaxation of the dark state

The kinetics of the photocycle of PYP and its mutants E46Q and E46A were investigated as a function of pH. E46 is the putative donor of the chromophore which becomes protonated in the I(2) intermediate. For E46Q we find that I(2) is in a pH-dependent equilibrium with its precursor I(1)' with a pK(a) of 8.15 and n = 1. From this result and from experiments with pH indicator dyes, we conclude that in the I(1)' to I(2) transition one proton is taken up from the external medium. The pK(a) of 8.15 is that of the surface-exposed chromophore in the equilibrium between I(1)' and I(2) and is close to that of the phenolate group of p-hydroxycinnamic acid. The pH-dependent I(1)'/I(2) equilibrium with associated H(+) uptake is reminiscent of the M(I)/M(II) equilibrium in the formation of the signaling state of rhodopsin. Well above this pK(a) no I(2) is formed and I(1)' returns in a pH-independent manner to the initial state P. The decay rate for the return to P via I(2) is between pH 4 and pH 8, exactly proportional to the hydroxide concentration (first order), and the deprotonation of the chromophore in this transition occurs by hydroxide uptake. Well above the pK(a) of 8.15 the apparent rate constant for the return to P is constant due to the branching from I(1)'. Complementary measurements with the pH indicator dye cresol red at pH 8.3 show that the remaining PYP molecules that still cycle via I(2) take up one proton in the formation of I(2). Together, these observations provide compelling evidence that during the photocycle the chromophore in E46Q is protonated and deprotonated from the external medium. For the yellow form of the mutant E46A the apparent rate constant for the return to P is also linear in [OH(-)] below about pH 8.3 and constant above about pH 9.5, with a pK(a) value of 8.8 for I(1)', suggesting a similar mechanism of chromophore protonation/deprotonation as in E46Q. For wild type qualitatively similar observations were made: the amplitude of I(2) decreased at alkaline pH, I(1)' and I(2) were in equilibrium, and I(1)' decayed together with the return to P. Chromophore hydrolysis prevented, however, an accurate determination of the pK(a) of I(1)'. We estimate that its value is above 11. The ground state P is in the dark in a pH-dependent equilibrium with a low-pH bleached form P(bl) with protonated chromophore. The pK(a) values for these equilibria are 4.8 and 7.9 for E46Q and E46A, respectively. When the pH is close to these pK(a)'s, the kinetics of the photocycle contains additional components in the millisecond time range. Using pH-jump stopped-flow experiments, we show that these contributions are due to the relaxation of the P/P(bl) equilibrium which is perturbed by the rapid decrease in the P concentration caused by the flash excitation of P. The condition for the occurrence of this effect is that the relaxation time of the P/P(bl) equilibrium is faster than the photocycle time.     

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.     

Lack of negative charge in the E46Q mutant of photoactive yellow protein prevents partial unfolding of the blue-shifted intermediate

The long-lived light-induced intermediate (pB) of the E46Q mutant (glutamic acid is replaced by glutamine at position 46) of photoactive yellow protein (PYP) has been investigated by NMR spectroscopy. The ground state of this mutant is very similar to that of wild-type PYP (WT), whereas the pB state, formed upon illumination, appears to be much more structured in E46Q than in WT. The differences are most striking in the N-terminal domain of the protein. In WT, the side-chain carboxylic group of E46 is known to donate its proton to the chromophore upon illumination. The absence of the carboxylic group near the chromophore in the E46Q mutant prohibits the formation of a negative charge at this position upon formation of pB. This prevents the partial unfolding of the mutant, as evidenced from NMR chemical shift comparison and proton/deuterium (H/D) exchange studies.     

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.     

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.     

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 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.     

Coupling of hydrogen bonding to chromophore conformation and function in photoactive yellow protein

To understand in atomic detail how a chromophore and a protein interact to sense light and send a biological signal, we are characterizing photoactive yellow protein (PYP), a water-soluble, 14 kDa blue-light receptor which undergoes a photocycle upon illumination. The active site residues glutamic acid 46, arginine 52, tyrosine 42, and threonine 50 form a hydrogen bond network with the anionic p-hydroxycinnamoyl cysteine 69 chromophore in the PYP ground state, suggesting an essential role for these residues for the maintenance of the chromophore's negative charge, the photocycle kinetics, the signaling mechanism, and the protein stability. Here, we describe the role of T50 and Y42 by use of site-specific mutants. T50 and Y42 are involved in fine-tuning the chromophore's absorption maximum. The high-resolution X-ray structures show that the hydrogen-bonding interactions between the protein and the chromophore are weakened in the mutants, leading to increased electron density on the chromophore's aromatic ring and consequently to a red shift of its absorption maximum from 446 nm to 457 and 458 nm in the mutants T50V and Y42F, respectively. Both mutants have slightly perturbed photocycle kinetics and, similar to the R52A mutant, are bleached more rapidly and recover more slowly than the wild type. The effect of pH on the kinetics is similar to wild-type PYP, suggesting that T50 and Y42 are not directly involved in any protonation or deprotonation events that control the speed of the light cycle. The unfolding energies, 26.8 and 25.1 kJ/mol for T50V and Y42F, respectively, are decreased when compared to that of the wild type (29.7 kJ/mol). In the mutant Y42F, the reduced protein stability gives rise to a second PYP population with an altered chromophore conformation as shown by UV/visible and FT Raman spectroscopy. The second chromophore conformation gives rise to a shoulder at 391 nm in the UV/visible absorption spectrum and indicates that the hydrogen bond between Y42 and the chromophore is crucial for the stabilization of the native chromophore and protein conformation. The two conformations in the Y42F mutant can be interconverted by chaotropic and kosmotropic agents, respectively, according to the Hofmeister series. The FT Raman spectra and the acid titration curves suggest that the 391 nm form of the chromophore is not fully protonated. The fluorescence quantum yield of the mutant Y42F is 1.8% and is increased by an order of magnitude when compared to the wild type.     

Binding, tuning and mechanical function of the 4-hydroxy-cinnamic acid chromophore in photoactive yellow protein.

The bacterial photoreceptor protein photoactive yellow protein (PYP) covalently binds the chromophore 4-hydroxy coumaric acid, tuning (spectral) characteristics of this cofactor. Here, we study this binding and tuning using a combination of pointmutations and chromophore analogs. In all photosensor proteins studied to date the covalent linkage of the chromophore to the apoprotein is dispensable for light-induced catalytic activation. We analyzed the functional importance of the covalent linkage using an isosteric chromophore-protein variant in which the cysteine is replaced by a glycine residue and the chromophore by thiomethyl-p-coumaric acid (TMpCA). The model compound TMpCA is shown to weakly complex with the C69G protein. This non-covalent binding results in considerable tuning of both the pKa and the color of the chromophore. The photoactivity of this system, however, was strongly impaired, making PYP the first known photosensor protein in which the covalent linkage of the chromophore is of paramount importance for the functional activity of the protein in vitro. We also studied the influence of chromophore analogs on the color and photocycle of PYP, not only in WT, but especially in the E46Q mutant, to test if effects from both chromophore and protein modifications are additive. When the E46Q protein binds the sinapinic acid chromophore, the color of the protein is effectively changed from yellow to orange. The altered charge distribution in this protein also results in a changed pKa value for chromophore protonation, and a strongly impaired photocycle. Both findings extend our knowledge of the photochemistry of PYP for signal generation.     

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.     

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.     

Transient exposure of hydrophobic surface in the photoactive yellow protein monitored with Nile Red

In this study we have investigated binding of the fluorescent hydrophobicity probe Nile Red to the photoactive yellow protein (PYP), to characterize the exposure and accessibility of hydrophobic surface upon formation of the signaling state of this photoreceptor protein. Binding of Nile Red, reflected by a large blue shift and increase in fluorescence quantum yield of the Nile Red emission, is observed exclusively when PYP resides in its signaling state. N-terminal truncation of the protein allows assignment of the region surrounding the chromophore as the site where Nile Red binds to PYP. We also observed a pH dependence of the affinity of Nile Red for pB, which we propose is caused by pH dependent differences of the structure of the signaling state. From a comparative analysis of the kinetics of Nile Red binding and transient absorption changes in the visible region we can conclude that protonation of the chromophore precedes the exposure of a hydrophobic surface near the chromophore binding site, upon formation of the signaling state. Furthermore, the data presented here favor the view that the signaling state is structurally heterogeneous.     

Molecular dynamics simulations of photoactive yellow protein (PYP) in three states of its photocycle: a comparison with X-ray and NMR data and analysis of the effects of Glu46 deprotonation and mutation

Photoactive yellow protein (PYP) is a prototype of the PAS domain superfamily of signaling proteins. The signaling process is coupled to a three-state photocycle. After the photoinduced trans-cis isomerization of the chromophore, 4-hydroxycinnamic acid (pCA), an early intermediate (pR) is formed, which proceeds to a second intermediate state (pB) on a sub-millisecond time scale. The signaling process is thought to be connected to the conformational changes upon the formation of pB and its recovery to the ground state (pG), but the exact signaling mechanism is not known. Experimental studies of PYP by solution NMR and X-ray crystallography suggest a very flexible protein backbone in the ground as well as in the signaling state. The relaxation from the pR to the pB state is accompanied by the protonation of the chromophore's phenoxyl group. This was found to be of crucial importance for the relaxation process. With the goal of gaining a better understanding of these experimental observations on an atomistic level, we performed five MD simulations on the three different states of PYP: a 1 ns simulation of PYP in its ground state [pG(MD)], a 1 ns simulation of the pR state [pR(MD)], a 2 ns simulation of the pR state with the chromophore protonated (pRprot), a 2 ns simulation of the pR state with Glu46 exchanged by Gln (pRGln) and a 2 ns simulation of PYP in its signaling state [pB(MD)]. Comparison of the pG simulation results with X-ray and NMR data, and with the results obtained for the pB simulation, confirmed the experimental observations of a rather flexible protein backbone and conformational changes during the recovery of the pG from the pB state. The conformational changes in the region around the chromophore pocket in the pR state were found to be crucially dependent on the strength of the Glu46-pCA hydrogen bond, which restricts the mobility of the chromophore in its unprotonated form considerably. Both the mutation of Glu46 with Gln and the protonation of the chromophore weaken this hydrogen bond, leading to an increased mobility of pCA and large structural changes in its surroundings. These changes, however, differ considerably during the pRGln and pRprot simulations, providing an atomistic explanation for the enhancement of the rate constant in the Gln46 mutant. Electronic supplementary material to this article is available at and is accessible for athorized users. Electronic Publication     

Early photocycle kinetic behavior of the E46A and Y42F mutants of photoactive yellow protein: femtosecond spectroscopy.

In the photoactive yellow protein, PYP, both Glu46 and Tyr42 form hydrogen bonds to the phenolic OH group of the p-hydroxycinnamoyl chromophore. Previous work on replacement of the carboxyl group of Glu46 by an amide group (Glu46Gln) has shown that changing the nature of this hydrogen bond has a minimal effect on the rate constant for the formation of the first intermediate (I(0)) and on the excited state lifetime, whereas the rate constants for the formation of the second (I(0)( not equal)) and third (I(1)) intermediates were increased by factors of approximately 30 and 5, respectively. In the present experiments, two additional mutants (Glu46Ala and Tyr42Phe) have been studied. These two mutants are shown to behave kinetically very similarly to one another. In both cases, the rate constant for I(0) formation is decreased by a factor of approximately 2, with little or no effect on the photochemical yield as a consequence of a compensating increase in the excited state lifetime. Although we are unable to resolve the rate constant for the formation of the second intermediate from that of the first intermediate, the rate constant for the formation of the third intermediate is increased by a factor of approximately 100. The structural implications of these results are discussed.     

A structural pathway for signaling in the E46Q mutant of photoactive yellow protein

In the bacterial photoreceptor photoactive yellow protein (PYP), absorption of blue light by its chromophore leads to a conformational change in the protein associated with differential signaling activity, as it executes a reversible photocycle. Time-resolved Laue crystallography allows structural snapshots (as short as 150 ps) of high crystallographic resolution (approximately 1.6 A) to be taken of a protein as it functions. Here, we analyze by singular value decomposition a comprehensive time-resolved crystallographic data set of the E46Q mutant of PYP throughout the photocycle spanning 10 ns-100 ms. We identify and refine the structures of five distinct intermediates and provide a plausible chemical kinetic mechanism for their inter conversion. A clear structural progression is visible in these intermediates, in which a signal generated at the chromophore propagates through a distinct structural pathway of conserved residues and results in structural changes near the N terminus, over 20 A distant from the chromophore.     

Helix formation is a dynamical bottleneck in the recovery reaction of Photoactive Yellow Protein

The bacterial sensor Photoactive Yellow Protein (PYP) signals the presence of blue light by undergoing a series of conformational changes. We present atomistic Parallel Tempering (Replica Exchange Molecular Dynamics) simulations of conformational changes occurring during the photo-cycle of PYP. First, we study the signaling state formation of PYP in detail. Our previous simulations have shown that the formation of the signaling state is characterized by the solvent exposure of both the chromophore and Glu46 (Vreede J, Crielaard W, Hellingwerf KJ, Bolhuis PG. Biophys J, 2005;8:3525-3535). Subsequent NMR results agreed with this prediction, but as these experiments were performed on an N-terminally truncated mutant, a simulation of this mutant would further substantiate our previous results. Here, we compare simulations of the truncated PYP to the NMR structures, as well as to the wild type predictions. This comparison also gives some insight into the role of the N-terminal domain of PYP, which restricts the movement of the chromophore binding pocket (CBP) in the wild type. Second, we report simulations of the recovery of the receptor state from the signaling state. While we did not observe complete refolding of the protein, we did observe transient interactions between residues of the CBP occurring when the chromophore is in a trans configuration. Using simulations that sample anomalous exposure of the chromophore in the receptor state, we were able to sample chromophore re-entry into its binding pocket. While the involved time scales prohibit drawing definitive conclusions even when using parallel tempering, we nevertheless propose that the formation of a helix in the CBP is essential for a successful recovery of the receptor state, and forms a kinetic barrier in this process.     

Proton movement and photointermediate kinetics in rhodopsin mutants

The role of ionizable amino acid side chains in the bovine rhodopsin activation mechanism was studied in mutants E134Q, E134R/R135E, H211F, and E122Q. All mutants exhibited bathorhodopsin stability on the 30 ns to 1 micros time scale similar to that of the wild type. Lumirhodopsin decay was also similar to that of the wild type except for the H211F mutant where early decay (20 micros) to a second form of lumirhodopsin was seen, followed by formation of an extremely long-lived Meta I(480) product (34 ms), an intermediate which forms to a much reduced extent, if at all, in dodecyl maltoside suspensions of wild-type rhodopsin. A smaller amount of a similar long-lived Meta I(480) product was seen after photolysis of E122Q, but E134Q and E134R/R135Q displayed kinetics much more similar to those of the wild type under these conditions (i.e., no Meta I(480) product). These results support the idea that specific interaction of His211 and Glu122 plays a significant role in deprotonation of the retinylidene Schiff base and receptor activation. Proton uptake measurements using bromcresol purple showed that E122Q was qualitatively similar to wild-type rhodopsin, with at least one proton being released during lumirhodopsin decay per Meta I(380) intermediate formed, followed by uptake of at least two protons per rhodopsin bleached on a time scale of tens of milliseconds. Different results were obtained for H211F, E134Q, and E134R/R135E, which all released approximately two protons per rhodopsin bleached. These results show that several ionizable groups besides the Schiff base imine are affected by the structural changes involved in rhodopsin activation. At least two proton uptake groups and probably at least one proton release group in addition to the Schiff base are present in rhodopsin.     

Monitoring the conformational changes of photoactivated rhodopsin from microseconds to seconds by transient fluorescence spectroscopy

The transient changes of the tryptophan fluorescence of bovine rhodopsin in ROS membranes were followed in time from 1 micros to 10 s after flash excitation of the photoreceptor. Up to about 100 micros the fluorescence did not change, suggesting that the tryptophan lifetimes in rhodopsin and the M(I) intermediate are similar. The fluorescence then decreases on the millisecond time scale with kinetics that match the rise of the M(II) state as measured on the same sample by the transient absorption increase at 360 nm. Both the sign and kinetics of the fluorescence change strongly suggest that it is due to an increase in energy transfer to the retinylidene chromophore caused by the increased spectral overlap in M(II). Calculation of the Forster radius of each tryptophan from the high-resolution crystal structure suggests that W265 and W126 are already completely quenched in the dark, whereas W161, W175, and W35 are located at distances from the retinal chromophore that are comparable to their Forster radii. The fluorescence from these residues is thus sensitive to an increase in energy transfer in M(II). Similar results were obtained at other temperatures and with monomeric rhodopsin in dodecyl maltoside micelles. A large light-induced transient fluorescence increase was observed with ROS membranes that were selectively labeled with Alexa594 at cysteine 316 in helix 8. Using transient absorption spectroscopy the kinetics of this structural change at the cytoplasmic surface was compared to the formation of the signaling state M(II) (360 nm) and to the kinetics of proton uptake as measured with the pH indicator dye bromocresol purple (605 nm). The fluorescence kinetics lags behind the deprotonation of the Schiff base. The proton uptake is even further delayed. These observations show that in ROS membranes (at pH 6) the sequence of events is Schiff base deprotonation, structural change, and proton uptake. From the temperature dependence of the kinetics we conclude that the Schiff base deprotonation and the transient fluorescence have comparable activation energies, whereas that of proton uptake is much smaller.     

The crystal structure of the R52Q mutant demonstrates a role for R52 in chromophore pKa regulation in photoactive yellow protein

Mutating arginine 52 to glutamine (R52Q) in photoactive yellow protein (PYP) increases the pK(a) of the chromophore by 1 pH unit. The structure of the R52Q PYP mutant was determined by X-ray crystallography and was compared to the structure of wild-type PYP to assess the role of R52 in pK(a) regulation. The essential differences between R52Q and the wild type were confined to the loop region containing the 52nd residue. While the hydrogen bonds involving the chromophore were unchanged by the mutation, removing the guanidino group generated a cavity near the chromophore; this cavity is occupied by two water molecules. In the wild type, R52 forms hydrogen bonds with T50 and Y98; these hydrogen bonds are lost in R52Q. Q52 is linked to Y98 by hydrogen bonding through the two water molecules. R52 acts as a lid on the chromophore binding pocket and controls the accessibility of the exterior solvent and the pK(a) of the chromophore. R52 is found to flip out during the formation of PYP(M). The result of this movement is quite similar to the altered structure of R52Q. Thus, we propose that conformational changes at R52 are partly responsible for pK(a) regulation during the photocycle.     

Protonation states and pH titration in the photocycle of photoactive yellow protein

Photoactive yellow protein (PYP) undergoes a light-driven cycle of color and protonation states that is part of a mechanism of bacterial phototaxis. This article concerns functionally important protonation states of PYP and the interactions that stabilize them, and changes in the protonation state during the photocycle. In particular, the chromophore pK(a) is known to be shifted down so that the chromophore is negatively charged in the ground state (dark state) even though it is buried in the protein, while nearby Glu46 has an unusually high pK(a). The photocycle involves changes of one or both of these protonation states. Calculations of pK(a) values and protonation states using a semi-macroscopic electrostatic model are presented for the wild-type and three mutants, in both the ground state and the bleached (I(2)) intermediate state. Calculations allowing multiple H-bonding arrangements around the chromophore also have been carried out. In addition, ground-state pK(a) values of the chromophore have been measured by UV-visible spectroscopy for the wild-type and the same three mutants. Because of the unusual protonation states and strong electrostatic interactions, PYP represents a severe test of the ability of theoretical models to yield correct calculations of electrostatic interactions in proteins. Good agreement between experiment and theory can be obtained for the ground state provided the protein interior is assumed to have a relatively low dielectric constant, but only partial agreement between theory and experiment is obtained for the bleached state. We also present a reinterpretation of previously published data on the pH-dependence of the recovery of the ground state from the bleached state. The new analysis implies a pK(a) value of 6.37 for Glu46 in the bleached state, which is consistent with other available experimental data, including data that only became available after this analysis. The new analysis suggests that signal transduction is modulated by the titration properties of the bleached state, which are in turn determined by electrostatic interactions. Overall, the results of this study provide a quantitative picture of the interactions responsible for the unusual protonation states of the chromophore and Glu46, and of protonation changes upon bleaching.     

Ligand binding reveals protonation events at the active site of cytochrome c oxidase; is the K-pathway used for the transfer of H(+) or OH(-)?

We have investigated the CO-recombination kinetics after flash photolysis of CO from the "half-reduced" cytochrome c oxidase as a function of pH. In addition, the reaction was investigated in mutant enzymes in which Lys(I-362) and Ser(I-299), located approximately in the middle of the K-pathway and near the enzyme surface, respectively, were modified. Laser-flash induced dissociation of CO is followed by rapid internal electron transfer from heme a(3) to a. At pH>7 this electron transfer is associated with proton release to the bulk solution (tau congruent with 1 ms at pH 8). Thus, the CO-recombination kinetics reflects protonation events at the catalytic site. In the wild-type enzyme, below pH approximately 7, the main component in the CO-recombination displayed a rate of approximately 20 s(-1). Above pH approximately 7, a slow CO-recombination component developed with a rate that decreased from approximately 8 s(-1) at pH 8 to approximately 1 s(-1) at pH 10. This slow component was not observed with KM(I-362), while with the SD(I-299)/SG(I-299) mutant enzymes at each pH it was slower than with the wild-type enzyme. The results are interpreted in terms of proton release from H(2)O in the catalytic site after CO dissociation, followed by OH(-) binding to the oxidized heme a(3). The CO-recombination kinetics is proposed to be determined by the protonation rate of OH(-) and not dissociation of OH(-), i.e. the K-pathway transfers protons and not OH(-). With the KM(I-362) mutant enzyme the proton is not released, i.e. OH(-) is not formed. With the SD(I-299)/SG(I-299) mutant enzymes the proton is released, but both the release and uptake are slowed by the mutations. During reaction of the reduced enzyme with O(2), the H(2)O at the binuclear center is most likely involved as a proton donor in the O-O cleavage reaction.