Blue-shifted Intermediate Research Articles

Kinetics of the E46Q mutant of photoactive yellow protein investigated by transient grating spectroscopy

To elucidate the role of internal proton transfer in the photodynamics of photoactive yellow protein (PYP), the photocycle of the Glu46Gln mutant of PYP (E46Q-PYP) is investigated by transient grating (TG) spectroscopy. Compared with wild-type PYP (wt-PYP), the first spectrally blue-shifted intermediate of E46Q-PYP is formed more slowly, which is consistent with the absence of direct protonation from Glu46 residue, if the parallel kinetic model for wt-PYP is invoked. The smaller conformational change in E46Q-PYP, as manifested by the smaller change in the diffusion coefficient, mainly arises from the relatively larger volume of the ground state E46Q-PYP than wt-PYP rather than from the smaller volume of the pB state.

Spectrally Silent Intermediates during the Photochemical Reactions of Salinibacter Sensory Rhodopsin I

Salinibacter sensory rhodopsin I (SrSRI) is a microbial rhodopsin discovered from the eubacterium Salinibacter ruber . It is thought to be a photoreceptor engaging the signal transductions for both positive and negative phototaxis. To elucidate the photoreactions of SrSRI in the presence and absence of chloride ions, we measured the refractive index change after the photoexcitation by the transient grating method. As a result, two spectrally silent processes were identified after the formation of M intermediate, and we named the spectrally identical intermediates M(1), M(2), and M(3). The enthalpy changes (ΔH) were estimated as ΔH = 136, 99, and 63 kJ/mol for K, M(1), and M(2) intermediates, respectively. The ΔH values were significantly decreased (36-55 kJ/mol) by the removal of chloride ions, suggesting their importance for structural changes of SrSRI. Volume expansions of SrSRI were observed on the spectrally silent steps (44 and 11 mL/mol). They may be related to the signaling process because blue-shifted intermediates of sensory rhodopsins are thought to be active state(s) for phototaxis.

Alpha‐retinals as Rhodopsin Chromophores—Preference for the 9‐Z Configuration and Partial Agonist Activity†

The visual pigment rhodopsin, the photosensory element of the rod photoreceptor cell in the vertebrate retina, shows in combination with an endogenous ligand, 11-Z retinal, an astonishing photochemical performance. It exhibits an unprecedented quantum yield (0.67) in a highly defined and ultrafast photoisomerization process. This triggers the conformational changes leading to the active state Meta(rhodopsin) II. Retinal is covalently bound to Lys-296 of the protein opsin in a protonated Schiff base. The resulting positive charge delocalization over the terminal part of the polyene chain of retinal creates a conjugation defect that upon photoexcitation moves to the opposite end of the polyene. Shortening the polyene as in 4,5-dehydro,5,6-dihydro (alpha), 5,6-dihydro or 7,8-dihydro-analogs might facilitate photoisomerization of a 9-Z and a 11-Z bond. Here we describe pigment analogs generated with bovine opsin and 11-Z or 9-Z 4,5-dehydro,5,6-dihydro-retinal that were further characterized by UV-Vis and FTIR spectroscopy. The preference of opsin for native 11-Z retinal over the 9-Z isomer is reversed in 4,5-dehydro,5,6-dihydro-retinal. 9-Z 4,5-dehydro,5,6-dihydro-retinal readily generated a photosensitive pigment. This modification has no effect on the quantum yield, but affects the Batho<-->blueshifted intermediate (BSI) equilibrium and leads to a strong decrease in the G-protein activation rate because of a downshift of the pK(a) of the Meta I<-->Meta II equilibrium.

The Solution Structure of a Transient Photoreceptor Intermediate: Δ25 Photoactive Yellow Protein

The N-terminally truncated variant of photoactive yellow protein (Delta25-PYP) undergoes a very similar photocycle as the corresponding wild-type protein (WT-PYP), although the lifetime of its light-illuminated (pB) state is much longer. This has allowed determination of the structure of both its dark- (pG) as well as its pB-state in solution by nuclear magnetic resonance (NMR) spectroscopy. The pG structure shows a well-defined fold, similar to WT-PYP and the X-ray structure of the pG state of Delta25-PYP. In the long-lived photocycle intermediate pB, the central beta sheet is still intact, as well as a small part of one alpha helix. The remainder of pB is unfolded and highly flexible, as evidenced by results from proton-deuterium exchange and NMR relaxation studies. Thus, the partially unfolded nature of the presumed signaling state of PYP in solution, as suggested previously, has now been structurally demonstrated.

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.

Assignment of Resonance Raman Spectrum of Photoactive Yellow Protein in Its Long-Lived Blue-Shifted Intermediate

Photoactive yellow protein (PYP) is a bacterial photoreceptor containing a 4-hydroxycinnamyl chromophore. We report the resonance Raman spectra for the long-lived blue-shifted intermediate of PYP whose chromophore is isotopically labeled with 13C at the carbonyl carbon atom or at the ring carbon atoms. Spectra have been also measured with PYP in D2O where the phenolic hydroxyl group of the chromophore is deuterated. All of the observed Raman bands are assigned on the basis of the observed isotope shifts and normal mode calculations using a density functional theory. The complete assignment provides a satisfactory framework for future investigations of the photocycle mechanism in PYP by vibrational spectroscopy.

Reaction of Neuronal Nitric-oxide Synthase with Oxygen at Low Temperature: EVIDENCE FOR REDUCTIVE ACTIVATION OF THE OXY-FERROUS COMPLEX BY TETRAHYDROBIOPTERIN

The reaction of reduced NO synthase (NOS) with molecular oxygen was studied at -30 degreesC. In the absence of substrate, the complex formed between ferrous NOS and O2 was sufficiently long lived for a precise spectroscopic characterization. This complex displayed similar spectral characteristics as the oxyferrous complex of cytochrome P450 (lambda max = 416.5 nm). It then decomposed to the ferric state. The oxidation of the flavin components was much slower and could be observed only at temperatures higher than -20 degreesC. In the presence of substrate (L-arginine), another, 12-nm blue-shifted, intermediate spectrum was formed. The breakdown of the latter species resulted in the production of Nomega-hydroxy-L-arginine in a stoichiometry of maximally 52% per NOS heme. This product formation took place also in the absence of the reductase domain of NOS. Both formation of the blue-shifted intermediate and of Nomega-hydroxy-L-arginine required the presence of tetrahydrobiopterin (BH4). We propose that the blue-shifted intermediate is the result of reductive activation of the oxygenated complex, and the electron is provided by BH4. These observations suggest that the reduction of the oxyferroheme complex may be the main function of BH4 in NOS catalysis.

Volume and Enthalpy Changes upon Photoexcitation of Bovine Rhodopsin Derived from Optoacoustic Studies by Using an Equilibrium between Bathorhodopsin and Blue‐Shifted Intermediate

AbstractA complex multistep model including an equilibrium was used for the first time to analyze data obtained by laser‐induced optoacoustic spectroscopy (LIOAS) with bovine rhodopsin in washed membranes (T = 5–32°C). The thermal equilibrium was between bathorhodopsin, batho, and the blue‐shifted intermediate (BSI), i.e., batho ⇄ BSI. This analysis considers a 14‐ns intermediate detected by LIOAS at 25°C (with similar decay activation parameters as those reported for BSI in solubilized rhodopsin). The energy content of lumirhodopsin (lumi) and the structural volume difference between lumi and rhodopsin were essentially the same (Elumi. = 80 kJ/mol and ΔVlumi–rho = 40 mL/mol) for both a model based on a simple batho → BSI → lumi sequence and one including the batho ⇄; BSI equilibrium. However, the structural volume changes for the individual steps batho → BSI and BSI → lumi were markedly changed by the introduction of the equilibrium. With the rate constant for the forward reaction batho → BSI for solubilized rhodopsin determined by Hug et al. (Hug, S.J.; Lewis, W.J.; Einterz, C.M.; Thorgeirsson, T.E.; Kliger, D.S. Biochemistry, 1990, 29, 1475–1485) the structural volume changes obtained with the equilibrium model are ΔVBSI‐batho = 79 mL/mol and ΔVlumi‐BSI = −44 mL/mol. The resulting batho → BSI expansion is larger than with the sequential model, and ΔVlumi‐BSI is now a contraction instead of an expansion. A small expansion of 5 mL/mol is derived for the formation of batho with both models. The estimated energy content of BSI is clearly higher than the value of lumi. The structural volume changes between batho, BSI, and lumi derived from the equilibrium model correlate better than those from the simple sequential model with the chromophore transition dipole moment changes determined in the literature using linear dichroism measurements. The structural volume changes are discussed in terms of the variations upon transformation of the chromophore–protein interactions.

Low-temperature trapping of early photointermediates of alpha-isorhodopsin.

Alpha-Isorhodopsin, an artificial visual pigment with a 9-cis-4,5-dehydro-5,6-dihydro(alpha)retinal chromophore, was photolyzed at low temperatures and absorption difference spectra were collected as the sample was warmed. A bathorhodopsin (Batho)-like intermediate absorbing at ca 495 nm was detected below 55 K,a blue-shifted intermediate (BSI)-like intermediate absorbing at ca 453 nm was observed when the temperature was raised to 60 K and a lumirhodopsin (Lumi)-like intermediate absorbing at ca 470 nm was found when the sample was warmed to 115 K. Photointermediates from this pigment were compared to those of native rhodopsin and 5,6-dihydroisorhodopsin. As in native rhodopsin, Batho is the first intermediate detected in alpha-isorhodopsin, though unlike native rhodopsin at low temperatures BSI is observed prior to Lumi formation. Alpha-Isohodopsin behaves similarly to 5,6-dihydroisorhodopsin, with the same early intermediates observed in both artificial visual pigments lacking the C5-C6 double bond. The transition temperature for BSI formation is higher in alpha-isorhodopsin, suggesting an interaction involving the chromophore ring in BSI formation. The transition temperature for Lumi formation is similar for these two pigments as well as for native rhodopsin, suggesting comparable changes in the protein environment in that transition.

Interactions of the beta-ionone ring with the protein in the visual pigment rhodopsin control the activation mechanism. An FTIR and fluorescence study on artificial vertebrate rhodopsins.

The photoreactions of rhodopsin regenerated with three 9-cis retinal analogs, modified at or in the vicinity of the beta-ionone ring (namely 5,6-epoxy, 7,8-diH, diethyl-acyclic) have been investigated by UV-vis and FTIR difference spectroscopy. In parallel, the ability to catalyze the GDP-->GTP exchange of G-protein (transducin) has been monitored by time-dependent fluorescence spectroscopy. The first photoproduct obtained with all three pigments at liquid nitrogen temperature is a blue-shifted intermediate (BSI), followed by a lumi-like intermediate at 170 K. For the 5,6-epoxy-ISO and 7,8-diH-ISO pigment we obtain two further intermediates similar to the META-I and META-II states of native RHO. For the diethyl-acyclic-ISO pigment only one further intermediate can be stabilized at 280 K. As compared to META-II the respective photoproduct exhibits striking differences. The latter two pigments have also been investigated in the solubilized lipid-free state (detergent: dodecyl maltoside) at 280 K. For the 5,6-epoxy-ISO pigment, the UV-vis, FTIR, and activation data agree with the formation of a META-II-like photoproduct (81% activation). Less META-II formation is observed for the 7,8-dihydro-ISO pigment in membranes (65% activation), but full formation in detergent (100% activation). Neither the membrane-bound nor the solubilized diethyl-acyclic-ISO pigment forms a META-II-like intermediate (18% and 0% activation, respectively). Therefore, we conclude that the substitution of the beta-ionone ring by two ethyl groups abolishes steric interactions with the protein, which are essential for META-II formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Absorption and photochemistry of sensory rhodopsin--I: pH effects.

Pyranine (8-hydroxyl-1,3,6-pyrene-trisulfonate) was used as a pH-probe to test whether there is a light-induced proton release to the bulk phase during the photochemical reaction cycle of sensory rhodopsin-I (SR-I). We conclude that the retinylidene Schiff-base proton is retained by SR-I-containing envelope vesicles during the SR-I photocycle under the conditions described here. Bacteriorhodopsin containing vesicles were used as a control to show that light-induced proton release can be observed under identical data acquisition parameters as those used for SR-I-containing vesicles. In addition, the effects of extravesicular pH on the absorption maximum (lambda max) and the SR-I photocycle were studied. SR-I properties are insensitive to pH in the range approximately 3 to approximately 8 with lambda max remaining at 587 nm. The lambda max shifts to 565 nm below pH 3.0 and to 552 nm at pH 10.8 with an apparent pKa of 8.5. Flash-induced absorbance changes of SR-I are described under neutral, alkaline and acidic conditions. The neutral, alkaline and acid SR-I forms each undergo similar photoreactions producing long-lived (> 500 ms decay half-time) blue-shifted intermediates. The UV/near-UV absorption of the photoproducts from neutral and alkaline SR-I indicate a deprotonated Schiff base, whereas acid SR-I produces a species with lambda max > 460 nm indicative of a protonated Schiff base.

Photolysis intermediates of human rhodopsin

Photochemical studies were conducted on human rhodopsin at 20 degrees C to characterize the intermediates which precede the formation of metarhodopsin II, the trigger for the enzyme cascade mechanism of visual transduction. Human rhodopsin was prepared from eyes which had previously been used for corneal donations. Time resolved absorption spectra collected from 10(-8) to 10(-6) s after photolysis of human rhodopsin in detergent suspensions displayed biexponential decay kinetics. The apparent lifetimes obtained from the data are 65 +/- 20 and 292 +/- 25 ns, almost a factor of 2 slower than the corresponding rates in bovine rhodopsin. The spectra can be fit well using a model in which human bathorhodopsin decays toward equilibrium with a blue-shifted intermediate (BSI) which then decays to lumirhodopsin. Spectra and kinetic rate constants were determined for all these intermediates using a global analysis which showed that the spectra of the human intermediates are remarkably similar to bovine intermediates. Microscopic rate constants derived from this model are 7.4 x 10(6) s-1 for bathorhodopsin decay and 7.5 x 10(6) s-1 and 4.6 x 10(6) s-1 for the forward and reverse reactions of BSI, respectively. Decay of lumirhodopsin to later intermediates was studied from 10(-6) to 10(-1) s after photolysis of rhodopsin in human disk membrane suspensions. The human metarhodopsin I in equilibrium metarhodopsin II equilibrium appears to be more forward shifted than in comparable bovine studies.

Nanosecond photolysis of rhodopsin: evidence for a new blue-shifted intermediate

Early photolysis intermediates of native bovine rhodopsin (RHO) are investigated by nanosecond laser photolysis near physiological temperature. Absorption difference spectra are collected after excitation with 477-, 532-, and 560-nm laser pulses of various energies and with 477-nm laser excitation at 5, 12, 17, 21, and 32 degrees C. The data are analyzed by using singular-value decomposition (SVD) and a global exponential fitting routine. Two rate constants associated with distinct spectral changes are observed during the time normally associated with the decay of bathorhodopsin to lumirhodopsin. Various models consistent with this observation are considered. A sequential model in which there is a reversible step between a bathorhodopsin intermediate and a new intermediate (BSI), which is blue-shifted relative to lumirhodopsin, is shown to best fit the data. The temperature dependence of the observed and calculated rate constants leads to linear Arrhenius plots. Extrapolation of the temperature dependence suggests that BSI should not be observable after rhodopsin photolysis at temperatures below -100 degrees C. The results are discussed with regard to the artificial visual pigments cis-5,6-dihydroisorhodopsin and 13-demethylrhodopsin. It is proposed that the rate of the BATHO to BSI transition is limited by the relaxation of the strained all-trans-retinal chromophore within a tight protein environment. The transition to LUMI involves chromophore relaxation concurrent with protein relaxation. While the first process is strongly affected by changes in the chromophore, the second transition seems to be determined more by protein relaxation.

Picosecond and nanosecond spectroscopies of visual pigments and other retinoid proteins.

Picosecond and nanosecond spectroscopic studies on the primary processes of chromoproteins with retinal (retinoid proteins) are reviewed. The retinoid proteins described here are a visual pigment rhodopsin and two analogues, bacteriorhodopsin and sensory rhodpsin. Bacteriorhodopsin and sensory rhodopsin are a light-driven proton pump and a photoreceptor of the phototaxis of halobacteria. There are the following common charac-teristics in their photo-induced reactions. (i) Photoreactions are initiated by photoisomerizations of retinals and followed by sequential thermal reactions. (ii) In the early stage red-shifted intermediates are formed. They are converted to more blue-shifted intermediates in the sequential reactions.