Complete assignment of the hydrogen out-of-plane wagging vibrations of bathorhodopsin: chromophore structure and energy storage in the primary photoproduct of vision

Resonance Raman vibrational spectra of the retinal chromophore in bathorhodopsin have been obtained after regenerating bovine visual pigments with an extensive series of 13C- and deuterium-labeled retinals. A low-temperature spinning cell technique was used to produce high-quality bathorhodopsin spectra exhibiting resolved hydrogen out-of-plane wagging vibrations at 838, 850, 858, 875, and 921 cm-1. The isotopic shifts and a normal coordinate analysis permit the assignment of these lines to the HC7 = C8H Bg, C14H, C12H, C10H, and C11H hydrogen out-of-plane wagging modes, respectively. The coupling constant between the C11H and C12H wags as well as the C12H wag force constant are unusually low compared to those of retinal model compounds. This quantitatively confirms the lack of coupling between the C11H and C12H wags and the low C12H wag vibrational frequency noted earlier by Eyring et al. [(1982) Biochemistry 21, 384]. The force constants for the C10H and C14H wags are also significantly below the values observed in model compounds. We suggest that the perturbed hydrogen out-of-plane wagging and C-C stretching force constants for the C10-C11 = C12-C13 region of the chromophore in bathorhodopsin result from electrostatic interactions with a charged protein residue. This interaction may also contribute to the 33 kcal/mol energy storage in bathorhodopsin.     

Resonance Raman studies of the primary photochemical event in visual pigments.

Resonance Raman multicomponent spectra of bovine rhodopsin, isorhodopsin, and bathorhodopsin have been obtained at low temperature. Application of the double beam "pump-probe" technique allows us to extract a complete bathorhodopsin spectrum from the mixture in both protonated and deuterated media. Our results show that the Schiff base of bathorhodopsin is fully protonated and that the extent of protonation is unaffected by its photochemical formation from either rhodopsin or isorhodopsin. The Raman spectrum of bathorhodopsin is significantly different than that of either parent pigment, thus supporting the notion that a geometric change in the chromophore is an important component of the primary photochemical event in vision. A normal mode analysis is carried out with particular attention devoted to the factors that determine the frequency of the C=N stretching vibration. We find that the increased frequency of this mode in protonated relative to unprotonated Schiff bases is due to coupling between C=N stretching and C=N-H bending motions, and the shift observed upon deuteration of the Schiff base can also be understood in these terms. Various models for the primary event are discussed in light of our experimental and theoretical results.     

Resonance Raman study of halorhodopsin photocycle kinetics, chromophore structure, and chloride-pumping mechanism.

Kinetic resonance Raman spectra of the HR520, HR640, and HR578 species in the halorhodopsin photocycle are obtained using time delays ranging from 5 microseconds to 10 ms in 0.3 M NO3-, 0.3 M Cl-, and 3 M Cl-. The Raman intensities are converted to absolute concentrations by using a conservation of molecules constraint. The simplest kinetic scheme that satisfactorily models the data is HR578-->HR520 in equilibrium with HR640-->HR578. The rate constant for the HR640-->HR578 transition increases with Cl- concentration, suggesting that Cl- is taken up between HR640 and HR578. The ratio of the forward to the reverse rate constants connecting HR520 and HR640 increases as the inverse of the Cl- concentration, suggesting that Cl- is released during the HR520-->HR640 step. The configuration about the C13 = C14 bond of the retinal chromophore in HR640 is examined by regenerating the protein with [12,14-2H2]retinal. The C12-2H + C14-2H rocking vibration for HR640 is observed at 943 cm-1, demonstrating that the chromophore is 13-cis. The changes in the resonance Raman spectrum of HR640 in response to 2H2O suspension indicates that the Schiff base linkage to the protein is protonated. None of the HR640 fingerprint vibrations shift significantly in 2H2O, suggesting that the Schiff base adopts a C = N anti configuration; this assignment is supported by the frequency of the C15-2H rocking mode (1002 cm-1). The 13-cis structure for the chromophore in HR640 requires that thermal isomerization back to all-trans occurs in the HR640-->HR578 transition. These structural and kinetic results are incorporated into a two-state C-T model for Cl- pumping.     

Resonance Raman study of the primary photochemistry of visual pigments. Hypsorhodopsin

We report here the first resonance Raman results of octopus hypsorhodopsin, a species formed photochemically at very low temperatures from visual pigments. A pump-probe technique was used to obtain Raman spectra from samples at 12 degrees K whose photostationary state mixtures were either hypsorhodopsin rich or hysorhodopsin poor. The data strongly suggest that the Schiff-base linkage between the chromophore of hysorhodopsin and apoprotein is protonated. Further, the results suggest that hypsorhodopsin's chromophore is in some torsionally distorted conformation, possibly having torsional departures from an all-trans isomeric form.     

Resonance Raman spectroscopic investigation of the light-harvesting chromophore in escherichia coli photolyase and Vibrio cholerae cryptochrome-1

Photolyases and cryptochromes are flavoproteins that belong to the class of blue-light photoreceptors. They usually bind two chromophores: flavin adenine dinucleotide (FAD), which forms the active site, and a light-harvesting pigment, which is a 5,10-methenyltetrahydrofolate polyglutamate (MTHF) in most cases. In Escherichia coli photolyase (EcPhr), the MTHF cofactor is present in substoichiometric amounts after purification, while in Vibrio cholerae cryptochrome-1 (VcCry1) the MTHF cofactor is bound more strongly and is present at stoichiometric levels after purification. In this paper, we have used resonance Raman spectroscopy to monitor the effect of loss of MTHF on the protein-FAD interactions in EcPhr and to probe the protein-MTHF interactions in both EcPhr and VcCry1. We find that removal of MTHF does not perturb protein-FAD interactions, suggesting that it may not affect the physicochemical properties of FAD in EcPhr. Our data demonstrate that the pteridine ring of MTHF in EcPhr has different interactions with the protein matrix than that of MTHF in VcCry1. Comparison to solution resonance Raman spectra of MTHF suggests that the carbonyl of its pteridine ring in EcPhr experiences stronger hydrogen bonding and a more polar environment than in VcCry1, but that hydrogen bonding to the pteridine ring amine hydrogens is stronger in VcCry-1. These differences in hydrogen bonding may account for the higher binding affinity of MTHF in VcCry1 compared to EcPhr.     

Resonance Raman characterization of the 7-ns photoproduct of (carbonmonoxy)hemoglobin: implications for hemoglobin dynamics

Resonance Raman spectra are reported for deoxyhemoglobin (deoxyHb) and the (carbonmonoxy)hemoglobin (HbCO) photoproduct Hb by use of 7-ns YAG laser pulses at wavelengths of 416 and 532 nm, where enhancement is observed for totally symmetric and nontotally symmetric modes, respectively. The frequencies of the porphyrin skeletal modes v10, v2, v19, v11, and v3 have been determined to be 1602, 1559, 1553, 1542, and 1466 cm-1 in Hb. These frequencies are 2-3 cm-1 lower than the corresponding frequencies for deoxyHb. The v19 and v11 frequencies are at the expected values for a Ct-N distance of 2.057 A, the known core size for a 6-coordinate high-spin FeII-porphyrin complex. The remaining frequencies, however, deviate from the core size correlations for these modes in the same direction as do those of deoxyHb, suggesting that the porphyrin ring is domed in both species. Thus, the heme structure is similar for deoxyHb and Hb but is slightly expanded in the latter. The expanded heme in Hb implies a restraint on the full out-of-plane displacement of the Fe atom, by an estimated approximately 0.1 A relative to deoxyHb. This could result from a residual interaction with the CO molecule if the latter remains held by the protein against the Fe atom, in a high-spin 6-coordinate complex. The available spectroscopic evidence suggests that such a complex may be stabilized at 4 K but is unlikely to persist at room temperature beyond the electronic relaxation (0.35 ps) of the electronically excited heme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of the chromophore of the third rhodopsin-like pigment of Halobacterium halobium and its photoproduct.

Halobacterium halobium contains at least three retinal-containing pigments: bacteriorhodopsin, halorhodopsin, and a third rhodopsin-like pigment (tR) absorbing at approximately 590 nm, tR590. Illumination of tR590 gives rise to a very long-lived blue absorbing photoproduct, tR370. Using high-performance liquid chromatography we show that the chromophore of tR590 is primarily all-trans retinal and its conversion by light to tR370 causes the chromophore to isomerize primarily to the 13-cis conformation. Irradiation of the tR370 gives rise to a transient photoproduct absorbing at approximately 520 nm that decays back to the initial pigment tR590. In addition to all-trans retinal, the apomembrane of tR can also combine with 13-cis retinal but not with the 9- or 11-cis isomers.     

Resonance Raman analysis of the Pr and Pfr forms of phytochrome

Resonance Raman vibrational spectra of the Pr and Pfr forms of oat phytochrome have been obtained at room temperature. When Pr is converted to Pfr, new bands appear in the C = C and C = N stretching region at 1622, 1599, and 1552 cm-1, indicating that a major structural change of the chromophore has occurred. The Pr to Pfr conversion results in an 11 cm-1 lowering of the N-H rocking band from 1323 to 1312 cm-1. Normal mode calculations correlate this frequency drop with a Z----E isomerization about the C15 = C16 bond. A line at 803 cm-1 in Pr is replaced by an unusually intense mode at 814 cm-1 in Pfr. Calculations on model tetrapyrrole chromophores suggest that these low-wavenumber modes are hydrogen out-of-plane (HOOP) wagging vibrations of the bridging C15 methine hydrogen and that both the intensity and frequency of the C15 HOOP mode are sensitive to the geometry around the C14-C15 and C15 = C16 bonds. The large intensity of the 814-cm-1 mode in Pfr indicates that the chromophore is highly distorted from planarity around the C15 methine bridge. If the Pr----Pfr conversion does involve a C15 = C16 Z----E isomerization, then the intensity of the C15 HOOP mode in Pfr argues that the chromophore has an E,anti conformation. On the basis of a comparison with the vibrational calculations, the low frequency (803 cm-1) and the reduced intensity of the C15 HOOP mode in Pr suggest that the chromophore in Pr adopts the C15-Z,syn conformation.     

Resonance Raman as a direct probe for the catalytic mechanism of molybdenum oxotransferases

 Recent studies of human sulfite oxidase and Rhodobacter sphaeroides DMSO reductase have demonstrated the ability of resonance Raman to probe in detail the coordination environment of the Mo active sites in oxotransferases via Mo=O, Mo-S(dithiolene), Mo-S(Cys) or Mo-O(Ser), dithiolene chelate ring and bound substrate vibrations. Furthermore, the ability to monitor the catalytically exchangeable oxo group via isotopic labeling affords direct mechanistic information and structures for the catalytically competent Mo(IV) and Mo(VI) species. The results clearly demonstrate that sulfite oxidase cycles between cis–di-oxo-Mo(VI) and mono-oxo-Mo(IV) states during catalytic turnover, whereas DMSO reductase cycles between mono-oxo-Mo(VI) and des-oxo-Mo(IV) states. In the case of DMSO reductase, ¹⁸O-labeling experiments have provided the first direct evidence for an oxygen atom transfer mechanism involving an Mo=O species. Of particular importance is that the active-site structures and detailed mechanism of DMSO reductase in solution, as determined by resonance Raman spectroscopy, are quite different to those reported or deduced in the three X-ray crystallographic studies of DMSO reductases from Rhodobacter species. Received: 16 June 1997 / Accepted: 20 August 1997     

Picosecond resonance Raman evidence for unrelaxed heme in the (carbonmonoxy)myoglobin photoproduct

An actively and passively mode-locked Nd:YAG laser, producing 30-ps pulses of 1-mJ energy at 532 nm, has been used to photolyze (carbonmonoxy)myoglobin (MbCO) and generate its resonance Raman spectrum, which was recorded with a vidicon multichannel analyzer. The photoproduct spectrum was obtained by subtraction of the MbCO spectrum, obtained at lower incident power levels. Comparison with the spectrum of deoxyMb, obtained with the same apparatus, revealed frequency downshifts of approximately 4 cm-1, for bands at 1604, 1554, and 1542 cm-1, which are identified with porphyrin skeletal modes v10, v19, and v11. These frequencies are known to correlate inversely with the core size of the porphyrin ring, and the shifts imply a larger core size for the photoproduct than for deoxyMb. Similar shifts have been observed for the (carbonmonoxy)hemoglobin (HbCO) photoproduct; in that case, the shifts persist for longer than 20 ns, whereas they are absent in the MbCO photoproduct spectrum within 7 ns of photolysis. The unrelaxed state of the heme group region is therefore suggested to be maintained by protein forces, which relax more rapidly for Mb than Hb. This may reflect a tighter coupling in Hb of the out-of-plane movement of the Fe atom with the proximal histidine-containing F helix.     

Structure of the retinal chromophore in sensory rhodopsin I from resonance Raman spectroscopy

Sensory rhodopsin I (SR-I) is a retinal-containing pigment which functions as a phototaxis receptor in Halobacterium halobium. We have obtained resonance Raman vibrational spectra of the native membrane-bound form of SR587 and used these data to determine the structure of its retinal prosthetic group. The similar frequencies and intensities of the skeletal fingerprint modes in SR587, bacteriorhodopsin (BR568), and halorhodopsin (HR578) as well as the position of the dideuterio rocking mode when SR-I is regenerated with 12,14-D2 retinal (915 cm-1) demonstrate that the retinal chromophore has an all-trans configuration. The shift of the C = N stretching mode from 1628 cm-1 in H2O to 1620 cm-1 in D2O demonstrates that the chromophore in SR587 is bound to the protein by a protonated Schiff base linkage. The small shift of the 1195 cm-1 C14-C15 stretching mode in D2O establishes that the protonated Schiff base bond has an anti configuration. The low value of the Schiff base stretching frequency together with its small 8 cm-1 shift in D2O indicates that the Schiff base proton is weakly hydrogen bonded to its protein counterion. This suggests that the red shift in the absorption maximum of SR-I (587 nm) compared with HR (578 nm) and BR (568 nm) is due to a reduction of the electrostatic interaction between the protonated Schiff base group and its protein counterion.     

Determination of retinal chromophore structure in bacteriorhodopsin with resonance Raman spectroscopy

The analysis of the vibrational spectrum of the retinal chromophore in bacteriorhodopsin with isotopic derivatives provides a powerful "structural dictionary" for the translation of vibrational frequencies and intensities into structural information. Of importance for the proton-pumping mechanism is the unambiguous determination of the configuration about the C13=C14 and C=N bonds, and the protonation state of the Schiff base nitrogen. Vibrational studies have shown that in light-adapted BR568 the Schiff base nitrogen is protonated and both the C13=C14 and C=N bonds are in a trans geometry. The formation of K625 involves the photochemical isomerization about only the C13=C14 bond which displaces the Schiff base proton into a different protein environment. Subsequent Schiff base deprotonation produces the M412 intermediate. Thermal reisomerization of the C13=C14 bond and reprotonation of the Schiff base occur in the M412------O640 transition, resetting the proton-pumping mechanism. The vibrational spectra can also be used to examine the conformation about the C--C single bonds. The frequency of the C14--C15 stretching vibration in BR568, K625, L550 and O640 argues that the C14--C15 conformation in these intermediates is s-trans. Conformational distortions of the chromophore have been identified in K625 and O640 through the observation of intense hydrogen out-of-plane wagging vibrations in the Raman spectra (see Fig. 2). These two intermediates are the direct products of chromophore isomerization. Thus it appears that following isomerization in a tight protein binding pocket, the chromophore cannot easily relax to a planar geometry. The analogous observation of intense hydrogen out-of-plane modes in the primary photoproduct in vision (Eyring et al., 1982) suggests that this may be a general phenomenon in protein-bound isomerizations. Future resonance Raman studies should provide even more details on how bacterio-opsin and retinal act in concert to produce an efficient light-energy convertor. Important unresolved questions involve the mechanism by which the protein catalyzes deprotonation of the L550 intermediate and the mechanism of the thermal conversion of M412 back to BR568. Also, it has been shown that under conditions of high ionic strength and/or low light intensity two protons are pumped per photocycle (Kuschmitz & Hess, 1981). How might this be accomplished?(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of Drosophila ninaG oxidoreductase in visual pigment chromophore biogenesis

We previously reported (Sarfare, S., Ahmad, S. T., Joyce, M. V., Boggess, B., and O'Tousa, J. E. (2005) J. Biol. Chem. 280, 11895-11901) that the Drosophila ninaG gene encodes an oxidoreductase involved in the biosynthesis of the (3S)-3-hydroxyretinal serving as chromophore for Rh1 rhodopsin and that ninaG mutant flies expressing Rh4 as the major opsin accumulate large amounts of a different retinoid. Here, we show that this unknown retinoid is 11-cis-3-hydroxyretinol. Reversed phase high performance liquid chromatography coupled with a photodiode array UV-visible absorbance detector and mass spectrometer revealed a major product eluting at a retention time, t(r), of 3.5 min with a lambda(max) of approximately 324 nm and with a base peak in the mass spectrum at m/z 285. These observations are identical with those of the 3-hydroxyretinol standard. The base peak in the electrospray ionization mass spectrum arises from the loss of a water molecule from the protonated molecule at m/z 303 because of fragmentation in the ion source. These results suggest that 11-cis-3-hydroxyretinol is an intermediate required for chromophore biogenesis in Drosophila. We further show that ninaG mutants fed on retinal as the sole source of vitamin A are able to synthesize 3-hydroxyretinoids. Thus, the NinaG oxidoreductase is not responsible for the initial hydroxylation of the retinal ring but rather acts in a subsequent step in chromophore production. These data are used to review chromophore biosynthesis and propose that NinaG acts in the conversion of (3R)-3-hydroxyretinol to the 3S enantiomer.     

Resonance Raman studies of sterically hindered cyanomet "strapped" hemes. Effects of ligand distortion and base tension on iron-carbon bond

We report resonance Raman studies of the iron-carbon bond stretching vibrations, nu(Fe-CN), in sterically hindered and unhindered heme (FeIII)-CN- complexes. The sterically hindred "strapped hemes" are equipped with a covalently linked 13-, 14-, or 15-atom hydrocarbon chain across one face of the heme; these are called FeSP-13, FeSP-14, and FeSP-15, respectively. These straps would presumably exert a sideway shearing strain to force the linear ligands (e.g., CN- and CO) to be tilted and/or bent. The shorter the chain length, the weaker the ligand binding affinity because of a greater steric hindrance. This study reveals that the nu(Fe-CN) frequency decreases as the chain length is decreased, in contrast with the CO complexes, where the nu(Fe-CO) frequency increases as the chain length is decreased. For the heme-CN- complexes (with N-methylimidazole as a base), the nu(Fe-CN) frequencies are: heme 5 (unhindered), 451 cm-1; FeSP-15, 447 cm-1; FeSP-14, 447 cm-1; FeSP-13, 445 cm-1. For the heme-CO complexes (with N-methylimidazole as a base), the nu(Fe-CO) frequencies are: heme 5, 495 cm-1; FeSP-15, 509 cm-1; FeSP-14, 512 cm-1; FeSP-13, 514 cm-1 (Yu, N.-T., E. A. Kerr, B. Ward, and C. K. Chang, 1983, Biochemistry, 22:4534-4540). We have also studied the cyanide complexes with three different bases (pyridine, N-methylimidazole and 1,2-dimethylimidazole), and found that the trans-effect of cyanide complex is different from that of CO complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resonance Raman microscopy of rod and cone photoreceptors

We have constructed a Raman microscope that has enabled us to obtain resonance Raman vibrational spectra from single photoreceptor cells. The laser beam which excites the Raman scattering is focused on the outer segment of the photoreceptor through the epiillumination system of a light microscope. Raman scattering from the visual pigment in the photoreceptor is collected by the objective and then dispersed onto a multichannel detector. High-quality spectra are recorded easily from individual outer segments that are 5 x 50 micrometer in size, and we have obtained spectra from cells as small as 1 x 10 micrometer. We have used the Raman microscope to study photostationary steady-state mixtures in pigments from toad (Bufo marinus) and goldfish (Carassius auratus) photoreceptors; these photoreceptors were frozen in glycerol glasses at 77 degrees K. Comparison of our toad red rod spectra with previously published spectra of bovine rod pigments demonstrates that the conformation of the chromophore in the first photointermediate, bathorhodopsin, is sensitive to variations in protein structure. We have also studied the first photointermediate in the goldfish rod photostationary steady-state. This bathoporphyropsin has a much lower ethylenic stretching frequency (1,507 cm-1) than that observed in the toad and bovine bathoproducts (approximately 1,535 cm-1). Preliminary results of our work on goldfish cone pigments are also reported. These are the first vibrational studies on the vertebrate photoreceptors responsible for color vision.     

Resonance Raman spectroscopy of iron-oxygen vibrations in hemerythrin

Resonance Raman spectra are reported for oxyhemerythrin and 15 anionic adducts of methemerythrin. All methemerythrin derivatives except sulfidomethemerythrin contain a Raman band near 510 cm^?1 which is assigned to an iron-oxygen stretching vibration. The effect of H₂¹⁸O on the frequency of this vibration was studied extensively. On the basis of the exchange results, the vibration is assigned to OH^?, H₂O, or a μ-oxo bridge between the irons.