Low-temperature Photoproducts Research Articles

Assignment of the Vibrational Modes of the Chromophores of Iodopsin and Bathoiodopsin: Low-Temperature Fourier Transform Infrared Spectroscopy of 13C- and 2H-Labeled Iodopsins

To investigate the chromophore structures of iodopsin and its low-temperature photoproducts, we have assigned their vibrational bands in the Fourier transform infrared (FTIR) spectra using iodopsin samples that were reconstituted with a series of (13)C- and deuterium-labeled retinals. The analyses of the vibrational bands in the fingerprint and hydrogen-out-of-plane (HOOP) regions indicated that the structure of the chromophores in the iodopsin system differs near their centers from those in the rhodopsin system. Compared to rhodopsin, the chromophore of the batho intermediate of iodopsin is twisted in the C(12) to C(14) regions but is more planar around C(11) region. The large amount of twisting was reduced by removing the chloride ion from the iodopsin, suggesting that this twisting hinders the relaxation of the torsion near C(11) necessary for the transition to the lumi intermediate and thus results in the thermal reversion of the batho intermediate back to the iodopsin. From the analyses of the C=NH and C=ND stretching bands, we conclude that the displacement of the Schiff base region upon photoisomerization of the chromophore is restricted, as is the case for rhodopsin. These results indicated that iodopsin's chromophore has a unique structure near its center and that this difference is enhanced by the binding of chloride nearby.

On the reaction mechanism of adduct formation in LOV domains of the plant blue-light receptor phototropin.

The blue-light sensitive photoreceptor, phototropin, is a flavoprotein which regulates the phototropism response of higher plants. The photoinduced triplet state and the photoreactivity of the flavin-mononucleotide (FMN) cofactor in two LOV domains of Avena sativa, Adiantum capillus-veneris, and Chlamydomonas reinhardtii phototropin have been studied by time-resolved electron paramagnetic resonance (EPR) and UV-vis spectroscopy at low temperatures (T < or = 80 K). Differences in the electronic structure of the FMN as reflected by altered zero-field splitting parameters of the triplet state could be correlated with changes in the amino acid composition of the binding pocket in wild-type LOV1 and LOV2 as well as in mutant LOV domains. Even at cryogenic temperatures, time-resolved EPR experiments indicate photoreactivity of the wild-type LOV domains, which was further characterized by UV-vis spectroscopy. Wild-type LOV1 and LOV2 were found to form an adduct between the FMN cofactor and the functional cysteine with a yield of 22% and 68%, respectively. The absorption maximum of the low-temperature photoproduct of wild-type LOV2 is red-shifted by about 15 nm as compared with the FMN C(4a)-cysteinyl adduct formed at room temperature. In light of these observations, we discuss a radical-pair reaction mechanism for the primary photoreaction in LOV domains.

Characterization of photocycle intermediates in crystalline photoactive yellow protein.

The photocycle in photoactive yellow protein (PYP) crystals was studied by single-crystal absorption spectroscopy with experimental setups for low-temperature and time-resolved measurements. Thin and flat PYP crystals, suitable for light absorption studies, were obtained using special crystallization conditions. Illumination of PYP crystals at 100 K led to the formation of a photostationary state, which includes at least one hypsochromic and one bathochromic photoproduct that resemble PYP(H) and PYP(B), respectively. The effect of temperature, light color and light pulse duration on the occupancy of these low-temperature photoproducts was determined and appeared similar to that observed in solution. At room temperature a blueshifted photocycle intermediate was identified that corresponds to the blueshifted state of PYP (pB). Kinetic studies show that the decay of this blueshifted intermediate is biphasic at -12 degrees C and 15-fold faster than that observed in solution at room temperature. These altered pB decay kinetics confirm a model that holds that the photocycle in crystals takes place in a shortcut version. In this version the key structural events of the photocycle, such as photoisomerization and reversible protonation of the chromophore, take place, but large conformational changes in the surrounding protein are limited by constraints imposed by the crystal lattice.

On the absorbance changes in the photocycle of the photoactive yellow protein: a quantum-chemical analysis.

Spectral changes in the photocycle of the photoactive yellow protein (PYP) are investigated by using ab initio multiconfigurational second-order perturbation theory at the available structures experimentally determined. Using the dark ground-state crystal structure [Genick, U. K., Soltis, S. M., Kuhn, P., Canestrelli, I. L. & Getzoff, E. D. (1998) Nature (London) 392, 206-209], the pipi* transition to the lowest excited state is related to the typical blue-light absorption observed at 446 nm. The different nature of the second excited state (npi*) is consistent with the alternative route detected at 395-nm excitation. The results suggest the low-temperature photoproduct PYP(HL) as the most plausible candidate for the assignment of the cryogenically trapped early intermediate (Genick et al.). We cannot establish, however, a successful correspondence between the theoretical spectrum for the nanosecond time-resolved x-ray structure [Perman, B., Srajer, V., Ren, Z., Teng, T., Pradervand, C., et al. (1998) Science 279, 1946-1950] and any of the spectroscopic photoproducts known up to date. It is fully confirmed that the colorless light-activated intermediate recorded by millisecond time-resolved crystallography [Genick, U. K., Borgstahl, G. E. O., Ng, K., Ren, Z., Pradervand, C., et al. (1997) Science 275, 1471-1475] is protonated, nicely matching the spectroscopic features of the photoproduct PYP(M). The overall contribution demonstrates that a combined analysis of high-level theoretical results and experimental data can be of great value to perform assignments of detected intermediates in a photocycle.

Polarized X-ray absorption spectroscopy of the low-temperature photoproduct of carbonmonoxy-myoglobin

Visible light can break the Fe—CO bond in Fe(II) carbonmonoxy-myoglobin (MbCO) giving an unligated product (Mb*) that is almost stable at T < 30 K. Fe K-edge polarized X-ray absorption spectra (P-XAS) of the photoproduct (T = 20 K) of an oriented single crystal (0.2 × 0.2 × 0.3 mm) of sperm whale MbCO (space group P21) have been collected. By rotating the crystal the X-ray photon polarization vector has been oriented almost parallel (with an angle α = 23°) or perpendicular (α = 86°) to the heme normal of each myoglobin molecule. The crystal was continuously illuminated by a white-light source during the data collection. The polarized data give novel information on the Fe-heme electronic/structural rearrangement following photolysis. The XANES (X-ray absorption near-edge structure) spectrum polarized in the direction close to the Fe—CO bond changes dramatically after photolysis, exhibiting a shift of ∼2 eV, due to electronic relaxation of empty states of pz symmetry, while more subtle changes are observed in the spectrum polarized along the heme plane, sensitive to the heme-plane geometry. Changes in the pre-edge region can be interpreted to provide insight into the electronic structure of the highest occupied and lowest unoccupied molecular orbitals (HOMO–LUMO) in the MbCO → Mb* photochemical reaction at low temperature.

Probing the heme axial ligation in the CO-sensing CooA protein with magnetic circular dichroism spectroscopy.

The combination of UV/visible/near-IR variable-temperature magnetic circular dichroism (VTMCD) and EPR spectroscopies has been used to investigate the spin states and axial ligation of the heme group in oxidized, reduced, and CO-bound reduced forms of the Rhodospirillum rubrum CO oxidation transcriptional activator protein (CooA) and its H77Y and C75S variants. The energy of the porphyrin(pi)-to-Fe(III) charge-transfer band (8930 cm(-)(1)) and the presence of cysteinate S-to-Fe(III) charge-transfer bands between 600 and 700 nm confirm cysteinate axial ligation to the low-spin Fe(III) hemes in oxidized wild-type and H77Y CooA. In contrast, the major component in the oxidized C75S variant is shown to be a low-spin Fe(III) heme with bis-histidine or histidine/amine axial ligation on the basis of the energy of the porphyrin(pi)-to-Fe(III) charge-transfer band (6240 cm(-)(1)) and the anisotropy of the EPR signal, g = 3.23, approximately 2.06, approximately 1.14. These results confirm Cys75 as the cysteinyl axial ligand in oxidized CooA, indicate that it is replaced as an axial ligand by a histidine in the C75S variant, and reveal the presence of a hitherto unidentified histidine or neutral nitrogen ligand trans to Cys75 in wild-type CooA. Evidence for a Cys75-to-His77 axial ligand switch on reduction of CooA comes from VTMCD studies of the reduced proteins. The VTMCD spectra of reduced wild-type and C75S CooA are dominated by bands characteristic of bis-histidine low-spin Fe(II) hemes, whereas the reduced H77Y variant is predominantly high-spin with MCD characteristics typical of a five-coordinate, histidine-ligated ferrous heme. VTMCD studies show that the CO-bound reduced forms of wild-type, H77Y, and C75S contain low-spin Fe(II) hemes and that the Fe-CO bonds can be photolytically cleaved at temperatures <50 K. Strong evidence that CO binding to the heme group in reduced CooA occurs with displacement of His77 comes from the VTMCD spectra of the low-temperature photoproducts of CO-bound reduced forms of wild-type, H77Y, and C75S CooA. The spectra are almost identical to each other and closely correspond to those of the low-temperature photoproducts of well characterized CO-bound ferrous hemes with His/CO axial ligation.

Stable Low-Temperature Photoproducts and Hole Burning of Green Fluorescent Protein (GFP)

We present low-temperature optical investigations down to 2 K of the photochemistry and hole burning of wild-type green fluorescent protein (wt-GFP). Three new photoproducts with absorption at 489, 502, and 510 nm (P489, P502, P510, respectively) are identified by fluorescence excitation and fluorescence spectroscopy. The data suggest that P502 is identical with the obligatory intermediate of the excited-state deprotonation cycle R-neq that exists only as a transient at room temperature. The second new product P510 is also associated with R-neq, whereas P489 is related to the equilibrated anionic form R-eq. An extended energy scheme accounting for these new species is presented. The ground-state energy barriers characterizing these products have been determined by temperature-dependent studies. Low-temperature persistent spectral hole burning in the red wing of the R-eq absorption has been demonstrated and can be used to characterize electron−phonon coupling.

Structural and Dynamic Properties of the Homodimeric Hemoglobin from Scapharca inaequivalvis Thr-72→Ile Mutant: Molecular Dynamics Simulation, Low Temperature Visible Absorption Spectroscopy, and Resonance Raman Spectroscopy Studies

Molecular dynamics simulations, low temperature visible absorption spectroscopy, and resonance Raman spectroscopy have been performed on a mutant of the Scapharca inaequivalvis homodimeric hemoglobin, where residue threonine 72, at the subunit interface, has been substituted by isoleucine. Molecular dynamics simulation indicates that in the Thr-72→Ile mutant several residues that have been shown to play a role in ligand binding fluctuate around orientations and distances similar to those observed in the x-ray structure of the CO derivative of the native hemoglobin, although the overall structure remains in the T state. Visible absorption spectroscopy data indicate that in the deoxy form the Soret band is less asymmetric in the mutant than in the native protein, suggesting a more planar heme structure; moreover, these data suggest a similar heme-solvent interaction in both the liganded and unliganded states of the mutant protein, at variance with that observed in the native protein. The “conformation sensitive” band III of the deoxy mutant protein is shifted to lower energy by >100 cm −1 with respect to the native one, about one-half of that observed in the low temperature photoproducts of both proteins, indicating a less polar or more hydrophobic heme environment. Resonance Raman spectroscopy data show a slight shift of the iron-proximal histidine stretching mode of the deoxy mutant toward lower frequency with respect to the native protein, which can be interpreted in terms of either a change in packing of the phenyl ring of Phe-97, as also observed from the simulation, or a loss of water in the heme pocket. In line with this latter interpretation, the number of water molecules that dynamically enters the intersubunit interface, as calculated by the molecular dynamics simulation, is lower in the mutant than in the native protein. The 10-ns photoproduct for the carbonmonoxy mutant derivative has a higher iron-proximal histidine stretching frequency than does the native protein. This suggests a subnanosecond relaxation that is slowed in the mutant, consistent with a stabilization of the R structure. Taken together, the molecular dynamics and the spectroscopic data indicate that the higher oxygen affinity displayed by the Thr-72→Ile mutant is mainly due to a local perturbation in the dimer interface that propagates to the heme region, perturbing the polarity of the heme environment and propionate interactions. These changes are consistent with a destabilization of the T state and a stabilization of the R state in the mutant relative to the native protein.

Iron L-Edge X-ray Absorption Spectroscopy of Myoglobin Complexes and Photolysis Products

We demonstrate the first application of L-edge X-ray absorption spectroscopy (XAS) to the electronic characterization of biological photolysis products. The experimental L-edge XAS spectra of deoxymyoglobin (deoxy Mb), oxymyoglobin (MbO2), carbonmonoxymyoglobin (MbCO), and the low-temperature photoproducts (Mb*CO and Mb*O2) are presented and compared to simulated spectra using a ligand field multiplet calculation. This analysis indicates that MbCO and MbO2 are both low spin and does not support some previous studies which suggest that MbO2 has an intermediate spin. Both photoproducts, Mb*CO and Mb*O2, are different from deoxy Mb in the FeII electronic structure. In addition, different low-temperature photolysis intermediates are suggested for MbCO and MbO2. The L-edge XAS spectra for FeIII in aquometmyoglobin (met Mb) and azidomet myoglobin (MbN3) provide a comparison of the ferrous versus ferric myoglobin species. Finally, the special advantages of using soft X-ray absorption spectroscopy for understandi...

Spectroscopic evidence for altered chromophore--protein interactions in low-temperature photoproducts of the visual pigment responsible for congenital night blindness.

The replacement of Gly90 by Asp in human rhodopsin causes congenital night blindness. It has been suggested that the molecular origin for the trait is an altered electrostatic environment of the protonated retinal Schiff base chromophore. We have investigated the corresponding recombinant bovine rhodopsin mutant G90D, as well as the related mutants E113A and G90D/E113A, using spectroscopy at low temperature. This allows the assessment of chromophore-protein interactions under conditions where conformational changes are mainly restricted to the retinal-binding site. Each of the mutant pigments formed bathorhodopsin- and isorhodopsin-like intermediates, but the concomitant visible absorption changes reflected differences in the electrostatic environment of the protonated Schiff base in each pigment. Fourier transform infrared-difference spectroscopy revealed effects on the chromophore fingerprint and hydrogen-out-of-plane vibrational modes, which were indicative of the removal of an electrostatic perturbation near C12 of the retinal chromophore in all three mutants. A comparison of the UV-visible and infrared-difference spectra of the mutant pigments strongly suggests that Glu113 is stably protonated in G90D. The corresponding carbonyl-stretching mode is assigned to a band at 1727 cm-1. In contrast to the case in native bathorhodopsin, the all-trans-retinal chromophores in the primary photoproducts of the mutant pigments are essentially relaxed. The peptide carbonyl vibrational changes in mutants G90D and G90D/ E113A suggest that this is due to a more flexible retinal-binding site. Therefore, the steric strain exerted on the chromophore in native bathorhodopsin may be caused by electrostatic forces that specifically involve glutamate 113.

Identification of Conformational Substates in Oxymyoglobin through the pH-Dependence of the Low-Temperature Photoproduct Yield

The effect of pH on the inner barrier to oxygen binding in myoglobin has been examined through a study of the pH-dependence of the low-temperature photoproduct yield for horse oxymyoglobin (MbO2). ...

Structural and Electronic Factors That Influence Oxygen Affinities: A Spectroscopic Comparison of Ferrous and Cobaltous Oxymyoglobin

Various structural and electronic factors that result in similar rates of oxygen association (kon) and differing rates of oxygen dissociation (koff) for ferrous (FeMb) and cobaltous (CoMb) myoglobin have been investigated. Similar values for kon indicate similar barriers to oxygen binding for CoMb and FeMb. Through optical spectroscopy, we have found that the stable quantum yields of photolysis for CoMbO2 (0.55 +/- 0.05) and FeMbO2 (0.50 +/- 0.05) at 10 K are the same. The X-ray absorption near edge spectra (XANES) of CoMb and FeMb reveal similar metal-heme displacements for the deoxy, oxy, and low temperature photoproduct states of CoMb and FeMb. Thus, similar barriers to ligand binding, indicated by similar kon's and photoproduct yields for CoMb and FeMb, correlate with the metal-heme displacements for the oxy, deoxy, and low temperature photoproduct states of CoMb and FeMb. Lower values of koff for FeMbO2 versus CoMbO2 imply different barriers to oxygen release for the two species. X-ray edge positions of CoMb and FeMb indicate a substantial transfer of electron density from the metal to the ligand upon oxygenation. The distribution of electron density throughout the M-O-O moiety differs for CoMbO2 and FeMbO2. Resonance Raman spectroscopy has demonstrated that the Co-O bond is weaker when compared to Fe-O [Tsubaki, M., & Yu, N. T. (1981) Proc. Natl. Acad. Sci., U.S.A. 78, 3581]. We have used photolyzed/unphotolyzed Fourier Transform Infrared (FTIR) difference spectra of CoMb16O2, CoMb18O2, FeMb16O2, and FeMb18O2 to show that the dioxygen stretching frequency, v(O-O), in CoMbO2 (approximately 1138 cm-1) is higher than FeMbO2 (approximately 1131 cm-1). The dioxygen stretching frequency in CoMbO2 is closer to that of heme protein models lacking a hydrogen bond to the distal histidine, suggesting that formation of the hydrogen bond in FeMbO2 provides a greater effect on the distribution of electron density throughout the Fe-O-O... HN moiety, potentially stabilizing a more ionic Fe-O-O bond. These findings demonstrate important electrostatic differences in the distal environments of CoMbO2 and FeMbO2, resulting in different barriers to oxygen release.

Investigations of optical line shapes and kinetic hole burning in myoglobin

We present the results of an extensive investigation of the optical line shapes of deoxymyoglobin (Mb), the ligand-bound form (MbCO), and the low-temperature photoproduct (Mb*). The thermal properties and the pH dependence of the Soret band and the near infrared band III (approximately 760 nm) are analyzed, taking into account the underlying vibrational properties of the absorption bands. The strong temperature dependence associated with the Soret band of MbCO and band III of Mb indicates significant coupling to low-frequency modes that may not be directly observed in the resonance Raman spectra. On the basis of analogous line-shape studies in a variety of heme systems, we assign the low-frequency coupling in MbCO to torsional motions of the CO molecule. The low-frequency mode coupled to band III (approximately 70 cm-1) is found to lie quite close to the value for the heme-doming motion (approximately 50 cm-1) calculated by using the kinetically determined value of the force constant (17 N/m). Significant inhomogeneous broadening in the Soret region of Mb and Mb* is found to be due to a "nonkinetic" coordinate that we associate with the orientation of the proximal histidine. A "kinetic" coordinate, associated with the equilibrium displacement of the iron atom from the porphyrin plane (a) is found to contribute to the inhomogeneous broadening of both the Soret band and band III. The relaxation of the heme as the system evolves from from Mb* to Mb is followed optically as a function of temperature, and a sharp transition temperature is found at 185 K. The blue shifts of the Soret band and band III as Mb* evolves to Mb are found to be nearly identical (delta v*ABS approximately 140 cm-1) and attributed to changes in the mean value of a between Mb* (a*0) and Mb (a0 = 0.45 A). A simple quadratic model for the coordinate coupling that simultaneously accounts for the observed shift, delta v*ABS, the low-temperature kinetics and the kinetic hole burning predicts a*0 = 0.2 +/- 0.05 A and EA = 16 +/- 2 kJ/mol for the room temperature Arrhenius barrier height at the heme. A simple quantitative method for the analysis of kinetic hole-burning experiments is also developed and applied to recent studies involving quaternary and subunit-specific hemoglobin structures.

Photoselection of conformational substates and the hyposchromic low-temperature photoproduct of rhodopsin

Photoselection of conformational substates and the hyposchromic low-temperature photoproduct of rhodopsin

Light induced proton transfer in dihydroxyanthraquinone as studied by photochemical hole burning

The low temperature photochemistry of dihydroxyanthraquinone in various host matrices has been studied via photochemical hole burning. It has been shown that the observed photochemistry is due to the rearrangement of protons involving both intra- and intermolecular hydrogen bonds. The model is supported by spectroscopic data on the low temperature photoproduct and by optical experiments on isotopically substituted and deprotonated samples. A brief discussion of the observed homogeneous linewidth links the optical data to possible radiationless deactivation processes (T1) or to optical dephasing processes (T2) on the time scale of several picoseconds.

ESR of the low temperature photoproducts in a diacetylene single crystal

We report ESR spectroscopy on the initial paramagnetic reaction products in the process of solid state photopolymerization. Low temperature UV-irradiation of the monomer diacetylene crystals produces a series of carbene ground state triplet centers. The different fine structure tensors and the hyperfine structure is analyzed.

Regeneration of Visual Pigments from their Low-temperature Photoproducts

Regeneration of Visual Pigments from their Low-temperature Photoproducts

Regeneration of visual pigments from their low-temperature photoproducts.

UNDER the above title Bridges has recently published an article which describes experiments said to be “in marked disagreement with the picture of the bleaching process” we have proposed1. We should like to comment briefly.

Regeneration of visual pigments from their low-temperature photoproducts.

Hubbard and Kropf's explanation of my results is based on the statement that the experiments were “performed in circumstances in which photochemical and thermal processes take place side by side”. The implication here is that irradiation converted the parent visual pigment to an orange photoproduct, and that this photoproduct was unstable and decayed to indicator yellow. This is not so. It was explicitly stated that the orange photoproduct produced by irradiating frog rhodopsin at − 5° C. was thermally stable.