Structural changes in lumirhodopsin and metarhodopsin I studied by their photoreactions at 77 K

The functional process of rhodopsin is initiated by cis-trans photoisomerization of the retinal chromophore. One of the primary intermediates, bathorhodopsin (Batho), is stable at 77 K, and structural changes in Batho are limited around the chromophore. Then, relaxation of Batho leads to helix opening at the cytoplasmic surface in metarhodopsin II (Meta II), which allows activation of a G protein transducin. Two intermediates, lumirhodopsin (Lumi) and metarhodopsin I (Meta I), appear between Batho and Meta II, and can be stabilized at 200 and 240 K, respectively. A photoaffinity labeling experiment reported that formation of Lumi accompanied flip-over of the beta-ionone ring of the retinal chromophore so that the ring portion was attached to Ala169 of helix IV [Borhan, B., Souto, M. L., Imai, H., Shichida, Y., and Nakanishi, K. (2000) Science 288, 2209-2212]. According to the crystal structure of bovine rhodopsin, the distance between the labeled C3 atom of the chromophore and Ala169 was >15 A [Palczewski, K., Kumasaka, T., Hori, T., Behnke, C. A., Motoshima, H., Fox, B. A., Le Trong, I., Teller, D. C., Okada, T., Stenkamp, R. E., Yamamoto, M., and Miyano, M. (2000) Science 289, 739-745]. These facts suggest that global protein structural changes such as helix motions take place in Lumi. In the study presented here, Lumi and Meta I are illuminated at 77 K, and protein structural changes are probed by Fourier transform infrared (FTIR) spectroscopy. We found that Lumi can be photoconverted to rhodopsin at 77 K from the IR spectral analysis of the photoproducts of Lumi. In contrast, more complex spectra were obtained for the photoproducts of Meta I at 77 K, implying that the protein structure of Meta I is considerably altered so as not to be reverted to the original state at 77 K. Thus, these photoreaction experiments with Lumi and Meta I at 77 K suggested the presence of global protein structural changes in the process between them. We concluded that the helix motions do not occur at Lumi, but at Meta I, and the flip-over of the beta-ionone ring reported by the photoaffinity labeling takes place through the specific reaction channel without a change in the global structure.     

Resonance Raman studies of bovine metarhodopsin I and metarhodopsin II

The resonance Raman spectra of bovine metarhodopsin I and metarhodopsin II have been measured. The spectra are compared with model chromophore resonance Raman data. It was found that metarhodopsin I is linked to opsin via a protonated Schiff base linkage, whereas metarhodopsin II is linked by an unprotonated Schiff base. A recent suggestion that the chromophore of metarhodopsin II is retinal is explicitly disproved. The chromophores of both metarhodopsins are found to have an essentially all-trans conformation. The basic mechanism for color regulation in both forms appears to be electron delocalization. The data tend to support the model of cis-trans isomerization as the primary mechanism for vision. Also, the conclusions and inferences of this work on energy uses and storage by rhodopsin in neural generation are discussed.     

Vibrational spectroscopy of excited electronic states in carotenoids in vivo. Picosecond time-resolved resonance Raman scattering.

The vibrational spectroscopy and population dynamics of excited singlet (2(1)Ag), excited triplet (3B u), and the ground (1Ag) electronic states of carotenoids in chromatophores of Chromatium vinosum (mainly spirilloxanthin and rhodopin) and of the same carotenoids in benzene solutions are examined by picosecond time-resolved resonance Raman scattering. Coherent Stokes Raman scattering from the ground states of carotenoids in chromatophores also is observed. Resonance Raman spectra of in vitro rhodopin and spirilloxanthin when compared with in vivo data demonstrate that scattering from spirilloxanthin dominates the in vivo spectrum. Comparisons of the time-dependent intensities of 2(1)Ag and 1Ag resonance Raman bands from both in vitro and in vivo carotenoids suggest that vibrationally excited levels in 1Ag are populated directly by the decay of the 2(1)Ag state and that these levels relax into a thermalized distribution in less than 50 ps. The appearance of asymmetrically broadened, ground-state resonance Raman bands supports this conclusion. Formation of the 3Bu state is observed for carotenoids in chromatophores, but not for in vitro spirilloxanthin indicating that the 3Bu state is formed by fission processes originating from the spatial organization of pigments within chromatophores. The rate at which the intensities of 2(1)Ag resonance Raman bands decay is faster for the carotenoids in vivo than for those in vitro thereby indicating that additional relaxation channels (e.g., energy transfer to bacteriochlorophylls) are present in the chromatophore. The similarity of the in vivo and in vitro 2(1)Ag resonance Raman spectra shows that no significant modifications in the vibronic coupling has been caused by the chromatophore environment.     

The retinal structure of channelrhodopsin-2 assessed by resonance Raman spectroscopy

Channelrhodopsin-2 mediates phototaxis in green algae by acting as a light-gated cation channel. As a result of this property, it is used as a novel optogenetic tool in neurophysiological applications. Structural information is still scant and we present here the first resonance Raman spectra of channelrhodopsin-2. Spectra of detergent solubilized and lipid-reconstituted protein were recorded under pre-resonant conditions to exclusively probe retinal in its electronic ground state. All-trans retinal was identified to be the favoured configuration of the chromophore but significant contributions of 13-cis were detected. Pre-illumination hardly changed the isomeric composition but small amounts of presumably 9-cis retinal were found in the light-adapted state. Spectral analysis suggested that the Schiff base proton is strongly hydrogen-bonded to a nearby water molecule.     

Picosecond dynamics of G-protein coupled receptor activation in rhodopsin from time-resolved UV resonance Raman spectroscopy

The protein response to retinal chromophore isomerization in the visual pigment rhodopsin is studied using picosecond time-resolved UV resonance Raman spectroscopy. High signal-to-noise Raman spectra are obtained using a 1 kHz Ti:Sapphire laser apparatus that provides <3 ps visible (466 nm) pump and UV (233 nm) probe pulses. When there is no time delay between the pump and probe events, tryptophan modes W18, W16, and W3 exhibit decreased Raman scattering intensity. At longer pump-probe time delays of +5 and +20 ps, both tryptophan (W18, W16, W3, and W1) and tyrosine (Y1 + 2xY16a, Y7a, Y8a) peak intensities drop by up to 3%. These intensity changes are attributed to decreased hydrophobicity in the microenvironment near at least one tryptophan and one tyrosine residue that likely arise from weakened interaction with the beta-ionone ring of the chromophore following cis-to-trans isomerization. Examination of the crystal structure suggests that W265 and Y268 are responsible for these signals. These UV Raman spectral changes are nearly identical to those observed for the rhodopsin-to-Meta I transition, implying that impulsively driven protein motion by the isomerizing chromophore during the 200 fs primary transition drives key structural changes that lead to protein activation.     

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)     

Studies of viral structure by Raman spectroscopy. I. R17 virus and R17 RNA

The laser-excited Raman spectrum of the RNA virus, R17, is shown to contain a large number of Raman lines assignable to scattering by vibrations of the nucleotide residues of RNA and the amino-acid residues of protein capsomers. The Raman lines from specific nucleotide vibrations in the phage are compared with their counterparts in the spectrum of protein-free RNA to suggest many similarities of RNA structure in the phage and protein-free states. However, the average configuration of guanine residues in the phage is apparently very different from that of protein-free RNA, suggesting that guanine plays an important role in RNA-protein interactions.     

Chromophore interactions in flavocytochrome c552: a resonance Raman investigation

Resonance Raman spectra with both Soret and visible excitation have been obtained for Chromatium flavocytochrome c552 and its isolated diheme subunit under varying conditions of pH and inhibitor binding. The spectra are generally consistent with previously established classification schemes for porphyrin ring vibrations. The presence of covalently bound flavin in the protein was apparent in the fluorescent background it produced and in flavin-mediated photoeffects observed in heme Raman spectra obtained at high laser power. No flavin modes were present in the Raman spectra, nor was any evidence of direct heme-flavin interaction found by using this technique; however, a systematic perturbation of heme B1g vibrational frequencies was found in the oxidized holoprotein. The heme vibrational frequencies of c552 are compared to those of the diheme peptide and of other c-type cytochromes. They are consistent with an interpretation that involves pH-dependent changes in axial ligation and treats the hemes and flavin as isolated chromphores communicating via protein-mediated interactions.     

Picosecond structural dynamics of myoglobin following photodissociation of carbon monoxide as revealed by ultraviolet time-resolved resonance Raman spectroscopy

Picosecond protein dynamics of myoglobin in response to structural changes in heme upon CO dissociation were observed in a site-specific fashion for the first time using time-resolved UV resonance Raman spectroscopy. Transient UV resonance Raman spectra showed several phases of intensity changes in both tryptophan and tyrosine Raman bands. Five picoseconds after dissociation, the W18, W16, and W3 bands of tryptophan residues and the Y8a band of tyrosine residues decreased in intensity, followed by recovery of the Y8a band intensity in hundreds of picoseconds and recovery of the tryptophan bands in nanoseconds. These spectral changes suggest that the change in heme structure impulsively drives concerted movement of the EF helical section and that rearrangements toward a deoxy structure occur in the heme vicinity and in the A helix within a time frame of sub-nanoseconds to nanoseconds.     

Antenna chlorophyll in photosynthetic membranes. A study by resonance Raman spectroscopy

Raman spectra of antenna chlorophyll a and chlorophyll b were selectively obtained from chloroplasts of green plants and from monocellular algae, using resonance enhancement in the respective Soret bands of these molecules, at 35 K. It is shown that:

Antenna chlorophyll a molecules occur in at least five discrete categories, distinguished by different extramolecular bonding of their 9-keto carbonyl groups.

These vibrational categories are probably identical in nature and number among the different organisms studied, but differ in their relative populations.

Chlorophyll b molecules occur in at least two different categories differing by the strength of the interactions of their 3-formyl C = 0 groups. These vibrational categories also appear as universal.

Most chlorophyll a and b molecules have their magnesium atoms bound to a single foreign ligand, whose nature may depend on the population considered.

Resonance Raman spectra of antenna structures, including those of organisms devoid of chlorophyll b, were compared to resonance Raman spectra of chlorophyll a and b in monomeric, oligomeric and hydrated polymeric states, at room temperature and at 35 K. No sizable amount of antenna chlorophyll a or b occurs as dry or hydrated oligomers, or polymers. The antenna molecules are thus necessarily bound to foreign molecules, probably proteins, through H-bonding on their formyl and/or keto carbonyl groups and through bonding of their magnesium atoms.     

Subpicosecond resonance Raman spectroscopy of carbonmonoxy- and oxyhemoglobin.

In this paper we present the resonance Raman spectrum of the carbonmonoxy- (HbCO) and oxyhemoglobin (HbO2) photointermediates on a 800-900 fs timescale. In the case of HbCO, the frequencies of the so-called core-size markers (1500-1650 cm-1) are characteristic of a deoxylike photoproduct in a high spin state (S = 2) with a partially domed heme. The spectrum of the HbO2 photointermediate, on the other hand, is different, and may be characteristic of an excited-state species. These results are discussed in terms of a reaction scheme previously presented by Petrich, J. W., C. Poyart, and J. L. Martin (1988. Biochemistry. 27:4049-4060) and compared with those obtained in the literature on a 30-40 ps timescale. In both molecules a distinct downshift of the v4 mode was observed with respect to the equilibrium value, which is indicative of an elevated temperature of the heme after photodissociation.     

Fourier transform resonance Raman spectroscopy of phytochrome.

The Pr and Pfr forms of phytochrome in H2O and D2O have been studied by Fourier transform resonance Raman spectroscopy with near-infrared excitation (1064 nm). It is demonstrated that this technique is a powerful method for analyzing the chromophore structures of photosensitive pigments. The high spectral quality allows discussion of vibrational assignments based on an empirical approach using previously published data obtained from model compounds. The reduction in intensity of a high-frequency band assigned to the ring-C/D methine bridge vibration is an indication for the non-coplanarity of the ring D in Pfr. The high intensity of a C-H out-of-plane vibration also supports this hypothesis. In Pr, a broad peak at approximately 1100 cm-1 is assigned to an out-of-plane vibration of a strongly hydrogen-bonded pyrrole C=NH+ group. It is missing in Pfr, suggesting deprotonation of the corresponding ring during the transformation from Pr to Pfr.     

Secondary and tertiary structure of the A-state of cytochrome c from resonance Raman spectroscopy.

Ferricytochrome c can be converted to the partially folded A-state at pH 2.2 in the presence of 1.5 M NaCl. The structure of the A-state has been studied in comparison with the native and unfolded states, using resonance Raman spectroscopy with visible and ultraviolet excitation wavelengths. Spectra obtained with 200 nm excitation show a decrease in amide II intensity consistent with loss of structure for the 50s and 70s helices. The 230-nm spectra contain information on vibrational modes of the single Trp 59 side chain and the four tyrosine side chains (Tyr 48, 67, 74, and 97). The Trp 59 modes indicate that the side chain remains in a hydrophobic environment but loses its tertiary hydrogen bond and is rotationally disordered. The tyrosine modes Y8b and Y9a show disruption of tertiary hydrogen bonding for the Tyr 48, 67, and 74 side chains. The high-wavenumber region of the 406.7-nm resonance Raman spectrum reveals a mixed spin heme iron atom, which arises from axial coordination to His 18 and a water molecule. The low-frequency spectral region reports on heme distortions and indicates a reduced degree of interaction between the heme and the polypeptide chain. A structural model for the A-state is proposed in which a folded protein subdomain, consisting of the heme and the N-terminal, C-terminal, and 60s helices, is stabilized through nonbonding interactions between helices and with the heme.     

Interaction of adriamycin with DNA as studied by resonance Raman spectroscopy.

Raman and resonance Raman spectra of the complex DNA-adriamycin in aqueous solution have been recorded and analysed. Calf thymus DNA was used and it is found that in the complex DNA-adriamycin the chromophore of adriamycin is intercalated in the GC sequences. The substituents on the rings give hydrogen bonding interactions with the base pairs above and below the intercalation site. It is suggested from the Raman and resonance Raman spectral modifications that the phenolic groups of the chromophore are involved in the drug-DNA intercalation, in addition to pi-pi, hydroxyl and amino group interactions.     

Tyrosine and tryptophan modification monitored by ultraviolet resonance Raman spectroscopy

Nitration of tyrosine with tetranitromethane shifts the tyrosine absorption spectrum and abolishes its 200 nm-excited resonance Raman spectrum. There is no detectable resonance Raman contribution from either reactants or products. Likewise, modification of tryptophan with 2-hydroxy-5-nitrobenzyl bromide (HNBB) shifts its absorption spectrum and abolishes its 218 nm-excited resonance Raman spectrum. In this case resonance Raman bands due to HNBB are seen, but are readily distinguishable from the tryptophan spectrum, can be computer-subtracted. When stellacyanin was treated with tetranitromethane the UV resonance Raman spectrum was greatly attenuated; quantitation of the 850 cm-1 tyrosine band intensity gave a value of 4.3 tyrosines modified out of the seven present in stellacyanin, in good agreement with an estimate of 4.7 from the absorption spectrum. For cytochrome c, the resonance Raman spectrum indicates that two out of the four tyrosines are modified by tetranitromethane treatment, consistent with the crystal structure, which shows two buried tyrosines and two at the protein surface. Treatment of stellacyanin with HNBB gave a reduction in the tryptophan spectrum, excited at 218 nm, consistent with one of the three tryptophans being modified. These modification procedures should be useful in distinguishing spectra of buried tyrosine and tryptophan residues from those at the surface.     

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.     

Photochemical cycle of bacteriorhodopsin studied by resonance Raman spectroscopy

Individual species of the photochemical cycle of bacteriorhodopsin, a retinal-protein complex of Halobacteria, were studied in aqueous suspensions of the "purple membrane" at room temperature by resonance Raman (RR) spectroscopy with flow systems. Two pronounced deuterium shifts were found in the RR spectra of the all-trans complex BR-570 in H2O-D2O suspensions. The first is ascribed to C=NH+ (C=ND+) stretching vibrations of the protonated Schiff base which links retinal to opsin. The second is assigned tentatively to an "X-H" ("X-D") bending mode, where "X" is an atom which carries an exchangeable proton. A RR spectrum of the 13-cis-retinal complex "BR-548" could be deduced from spectra of the dark-adapted purple membrane. The RR spectrum of the M-412 intermediate was monitored in a double-beam pump-probe experiment. The main vibrational features of the intermediate M' in the reaction M-412 in equilibrium hv M' leads to delta BR-570 could be deduced from a photostationary mixture of M-412 and M'. Difference procedures were applied to obtain RR spectra of the L-550 intermediate and of two new long-lived species, R1'-590 and R2-550. From kinetic data it is suggested that T1'-590 links the proton-translocating cycle to the "13-cis" cycle of BR-548. The protonation and isomeric states of the different species are discussed in light of the new spectroscopic and kinetic data. It is found that conformational changes during the photochemical cycle play an important role.     

Linking conformation change to hemoglobin activation via chain-selective time-resolved resonance Raman spectroscopy of protoheme/mesoheme hybrids

Time-resolved resonance Raman (RR) spectra are reported for hemoglobin (Hb) tetramers, in which the α and β chains are selectively substituted with mesoheme. The Soret absorption band shift in mesoheme relative to protoheme permits chain-selective recording of heme RR spectra. The evolution of these spectra following HbCO photolysis shows that the geminate recombination rates and the yields are the same for the two chains, consistent with recent results on ¹⁵N-heme isotopomer hybrids. The spectra also reveal systematic shifts in the deoxyheme ν ₄ and ν _Fe–His RR bands, which are anticorrelated. These shifts are resolved for the successive intermediates in the protein structure, which have previously been determined from time-resolved UV RR spectra. Both chains show Fe–His bond compression in the immediate photoproduct, which relaxes during the formation of the first intermediate, R_deoxy (0.07 μs), in which the proximal F-helix is proposed to move away from the heme. Subsequently, the Fe–His bond weakens, more so for the α chains than for the β chains. The weakening is gradual for the β chains, but is abrupt for the α chains, coinciding with completion of the R–T quaternary transition, at 20 μs. Since the transition from fast- to slow-rebinding Hb also occurs at 20 μs, the drop in the α chain ν _Fe–His supports the localization of ligation restraint to tension in the Fe–His bond, at least in the α chains. The mechanism is more complex in the β chains.