Procatalytic ligand strain. Ionization and perturbation of 8-nitroxanthine at the urate oxidase active site

The binding of the inhibitor 8-nitroxanthine to urate oxidase has been investigated by Raman and UV-visible absorption spectroscopy. The absorption maximum of 8-nitroxanthine shifts from 380 to 400 nm upon binding to the enzyme, demonstrating that the electronic structure of the ligand is perturbed. It has been proposed that oxidation of the substrate urate by urate oxidase is facilitated by formation of the substrate dianion at the enzyme active site, and Raman spectra of urate oxidase-bound 8-nitroxanthine suggest that both the dianionic and monoanionic forms of the ligand are bound to the enzyme under conditions where in solution the monoanion is present exclusively. The C4-C5 stretching frequency appears as a relatively isolated vibrational mode in 8-nitroxanthine whose frequency shifts according to the protonation state of the purine ring. Identification of the C4-C5 stretching mode was confirmed using [4-(13)C]-8-nitroxanthine and ab initio calculation of the vibrational modes. Two peaks corresponding to the C4-C5 stretching mode were evident in spectra of enzyme-bound 8-nitroxanthine, at 1541 and 1486 cm(-)(1). The higher frequency peak was assigned to monoanionic 8-nitroxanthine, and the low-frequency peak was assigned to dianionic 8-nitroxanthine. The C4-C5 stretching frequency for free monoanionic 8-nitroxanthine was at 1545 cm(-)(1), indicating that the enzyme polarizes that bond when the ligand is bound. The C4-C5 stretching frequency in dianionic 8-nitroxanthine is also shifted by 4 cm(-)(1) to lower frequency upon binding. For 8-nitroxanthine free in solution, the C4-C5 stretching frequency shifts to lower frequency upon deprotonation, and the absorption maximum in the UV-visible spectrum shifts to higher wavelength. The spectral shifts observed upon binding of 8-nitroxanthine to urate oxidase are consistent with increased anionic character of the ligand, which is expected to promote catalysis in the reaction with the natural substrate urate. In the Raman spectra of 8-nitroxanthine bound to the F179A, F179Y, and K9M mutant proteins, the C4-C5 stretching frequency was not perturbed from its position for the unbound ligand. Both V(max) and V/K were decreased in the mutant enzymes, demonstrating a correlation between the interaction that perturbs the C4-C5 stretching frequency and the catalytic activity of the enzyme. It is suggested that hydrogen-bonding interactions that lead to precise positioning and deprotonation of the substrate are perturbed by the mutations.     

Determination of molecular changes in soft tissues under strain using laser Raman microscopy

The paper presents a non-contact technique to examine the molecular changes in a collagen fibre subjected to in vitro axial tension. Laser Raman microscopy was employed to monitor the vibrational changes in specific assignments of the Raman spectrum of collagen. Results were presented in the form of Raman wavenumber shift as a function of applied tensile strain. Two distinct responses were observed depending on whether the vibrations were axial to, or normal to, the collagen backbone. The former response produced a decrease in wavenumber values, indicating tension, whereas the latter produced an increase, indicating compression. The rate of wavenumber shift with applied strain was non-linear in form, with a marked increase at higher levels of applied strain, for example, a strain 4% in the case of axial vibrations. This technique can prove to be a powerful tool for examining deformation at the molecular level in collagenous tissues.     

Nanosecond retinal structure changes in K-590 during the room-temperature bacteriorhodopsin photocycle: picosecond time-resolved coherent anti-stokes Raman spectroscopy.

Time-resolved vibrational spectra are used to elucidate the structural changes in the retinal chromophore within the K-590 intermediate that precedes the formation of the L-550 intermediate in the room-temperature (RT) bacteriorhodopsin (BR) photocycle. Measured by picosecond time-resolved coherent anti-Stokes Raman scattering (PTR/CARS), these vibrational data are recorded within the 750 cm-1 to 1720 cm-1 spectral region and with time delays of 50-260 ns after the RT/BR photocycle is optically initiated by pulsed (< 3 ps, 1.75 nJ) excitation. Although K-590 remains structurally unchanged throughout the 50-ps to 1-ns time interval, distinct structural changes do appear over the 1-ns to 260-ns period. Specifically, comparisons of the 50-ps PTR/CARS spectra with those recorded with time delays of 1 ns to 260 ns reveal 1) three types of changes in the hydrogen-out-of-plane (HOOP) region: the appearance of a strong, new feature at 984 cm-1; intensity decreases for the bands at 957 cm-1, 952 cm-1, and 939 cm-1; and small changes intensity and/or frequency of bands at 855 cm-1 and 805 cm-1; and 2) two types of changes in the C-C stretching region: the intensity increase in the band at 1196 cm-1 and small intensity changes and/or frequency shifts for bands at 1300 cm-1 and 1362 cm-1. No changes are observed in the C = C stretching region, and no bands assignable to the Schiff base stretching mode (C = NH+) mode are found in any of the PTR/CARS spectra assignable to K-590. These PTR/CARS data are used, together with vibrational mode assignments derived from previous work, to characterize the retinal structural changes in K-590 as it evolves from its 3.5-ps formation (ps/K-590) through the nanosecond time regime (ns/K-590) that precedes the formation of L-550. The PTR/CARS data suggest that changes in the torsional modes near the C14-C15 = N bonds are directly associated with the appearance of ns/K-590, and perhaps with the KL intermediate proposed in earlier studies. These vibrational data can be primarily interpreted in terms of the degree of twisting of the C14-C15 retinal bond. Such twisting may be accompanied by changes in the adjacent protein. Other smaller, but nonetheless clear, spectral changes indicate that alterations along the retinal polyene chain also occur. The changes in the retinal structure are preliminary to the deprotonation of the Schiff base nitrogen during the formation of M-412. The time constant for the ps/ns K-590 transformation is estimated from the amplitude change of four vibrational bands in the HOOP region to be 40-70 ns.     

Molecular structure and effects of intermolecular hydrogen bonding on the vibrational spectrum of trifluorothymine, an antitumor and antiviral agent

In the present work, the experimental and the theoretical vibrational spectra of trifluorothymine were investigated. The FT-IR (400-4000?cm(-1)) and μ-Raman spectra (100-4000?cm(-1)) of trifluorothymine in the solid phase were recorded. The geometric parameters (bond lengths and bond angles) and vibrational frequencies of the title molecule in the ground state were calculated using ab initio Hartree-Fock (HF) method and density functional theory (B3LYP) method with the 6-31++G(d,p) and 6-311++G(d,p) basis sets for the first time. The optimized geometric parameters and the theoretical vibrational frequencies were found to be in good agreement with the corresponding experimental data and with results found in the literature. Vibrational frequencies were assigned based on the potential energy distribution using the VEDA 4 program. The dimeric form of trifluorothymine was also simulated to evaluate the effect of intermolecular hydrogen bonding on the vibrational frequencies. It was observed that the stretching modes shifted to lower frequencies, while the in-plane and out-of-plane bending modes shifted to higher frequencies due to the intermolecular N-H?O hydrogen bonds.     

Density functional and Møller–Plesset studies of cyclobutanone⋯HF and ⋯HCl complexes

The molecular structure (hydrogen bonding, bond distances and angles), dipole moment and vibrational spectroscopic data [vibrational frequencies, IR and vibrational circular dichroism (VCD)] of cyclobutanone?HX (X?=?F, Cl) complexes were calculated using density functional theory (DFT) and second order Møller–Plesset perturbation theory (MP2) with basis sets ranging from 6–311G, 6–311G^**, 6–311 + + G^**. The theoretical results are discussed mainly in terms of comparisons with available experimental data. For geometric data, good agreement between theory and experiment is obtained for the MP2 and B3LYP levels with basis sets including diffuse functions. Surface potential energy calculations were carried out with scanning HCl and HF near the oxygen atom. The nonlinear hydrogen bonds of 1.81 Å and 175° for HCl and 1.71 Å and 161° for HF were calculated. In these complexes the C=O and H–X bonds participating in the hydrogen bond are elongated, while others bonds are compressed. The calculated vibrational spectra were interpreted and the band assignments reported are in excellent agreement with experimental IR spectra. The C=O stretching vibrational frequencies of the complexes show red shifts with respect to cyclobutanone.     

Vibrational frequency shifts as a probe of hydrogen bonds: thermal expansion and glass transition of myoglobin in mixed solvents

The contribution of hydrogen bonds to protein-solvent interactions and their impact on structural flexibility and dynamics of myoglobin are discussed. The shift of vibrational peak frequencies with the temperature of myoglobin in sucrose/water and glycerol/water solutions is used to probe the expansion of the hydrogen bond network. We observe a characteristic change in the temperature slope of the O–H stretching frequency at the glass transition which correlates with the discontinuity of the thermal expansion coefficient. The temperature-difference spectra of the amide bands show the same tendency, indicating that stronger hydrogen bonding in the bulk affects the main-chain solvent interactions in parallel. However, the hydrogen bond strength decreases relative to the bulk solvent with increasing cosolvent concentration near the protein surface, which suggests preferential hydration. Weaker and/or fewer hydrogen bonds are observed at low degrees of hydration. The central O–H stretching frequency of protein hydration water is red-shifted by 40 cm^–1 relative to the bulk. The shift increases towards lower temperatures, consistent with contraction and increasing strength of the protein-water bonds. The temperature slope shows a discontinuity near 180 K. The contraction of the network has reached a critical limit which leads to frozen-in structures. This effect may represent the molecular mechanism underlying the dynamic transition observed for the mean square displacements of the protein atoms and the heme iron of myoglobin. Received: 10 July 1996 / Accepted: 10 April 1997     

Characterization of hydroxyl groups of highly crystalline β-chitin under static tension detected by FT-IR

The different behavior of the OH groups of a highly crystalline β-chitin from Lamellibrachia sp. was revealed by FT-IR spectroscopy with static tension applied in the direction of the chain. Both OH groups shifted to higher wavenumbers under the tension, possibly because the force constant of the OH vibration intensified due to the erosion of hydrogen bonds, but the shift in the OH peak occurring at 3435 cm⁻¹ was 14% greater than that of the peak at 3470 cm⁻¹. The band at 3435 cm⁻¹ was assigned to O(3)H and that at 3470 cm⁻¹ to O(6)H, because the intramolecular hydrogen bond of O(3)H?O(5) occurs along the chitin chain's backbone and is likely to be more affected under tensile stress as a load carrier, than O(6)H?O(7)C, which is an intermolecular hydrogen bond located in a branch of the chain. Computation of an IR spectrum under stretching was conducted and supported the assignment of the two OH groups.     

Low-frequency vibrations in resonance Raman spectra of horse heart myoglobin. Iron-ligand and iron-nitrogen vibrational modes.

The low-frequency regions (150--700 cm-1) of resonance Raman (RR) spectra of various complexes of oxidized and reduced horse heart myoglobin were examined by use of 441.6-nm excitation. In this frequency range, RR spectra show 10 bands common to all myoglobin derivatives (numbered here for convenience from I to X). Relative intensities of bands IV, V, and X constitute good indicators of the doming state of the heme and, consequently, of the spin state of the iron atom. An additional band is present for several complexes (fluorometmyoglobin, hydroxymetmyoglobin, azidometmyoglobin, and oxymyoglobin). Isotopic substitutions on the exogenous ligands and of the iron atom (56Fe leads to 54Fe) allow us to assign these additional lines to the stretching vibrations of the Fe-sixth ligand bond. Similarly, bands II are assigned to stretching vibrations of the Fe-N-(pyrrole) bonds. An assignment of bands VI to stretching vibrations of the Fe-Nepsilon(proximal histidine) bonds is also proposed. Mechanisms for the resonance enhancement of the main low-frequency bands are discussed on the basis of the excitation profiles and of the dispersion curves for depolarization ratios obtained for fluorometmyoglobin and hydroxymetmyoglobin.     

Vibrational Stark effects calibrate the sensitivity of vibrational probes for electric fields in proteins

Infrared spectroscopy is widely used to probe local environments and dynamics in proteins. The introduction of a unique vibration at a specific site of a protein or more complex assembly offers many advantages over observing the spectra of an unmodified protein. We have previously shown that infrared frequency shifts in proteins can arise from differences in the local electric field at the probe vibration. Thus, vibrational frequencies can be used to map electric fields in proteins at many sites or to measure the change in electric field due to a perturbation. The Stark tuning rate gives the sensitivity of a vibrational frequency to an electric field, and for it to be useful, the Stark tuning rate should be as large as possible. Vibrational Stark effect spectroscopy provides a direct measurement of the Stark tuning rate and allows a quantitative interpretation of frequency shifts. We present vibrational Stark spectra of several bond types, extending our work on nitriles and carbonyls and characterizing four additional bond types (carbon-fluorine, carbon-deuterium, azide, and nitro bonds) that are potential probes for electric fields in proteins. The measured Stark tuning rates, peak positions, and extinction coefficients provide the primary information needed to design amino acid analogues or labels to act as probes of local environments in proteins.     

Molecular changes during tensile deformation of single wood fibers followed by Raman microscopy

Raman spectra were acquired in situ during tensile straining of mechanically isolated fibers of spruce latewood. Stress-strain curves were evaluated along with band positions and intensities to monitor molecular changes due to deformation. Strong correlations (r = 0.99) were found between the shift of the band at 1097 cm(-1) corresponding to the stretching of the cellulose ring structure and the applied stress and strain. High overall shifts (-6.5 cm(-1)) and shift rates (-6.1 cm(-1)/GPa) were observed. After the fiber failed, the band was found on its original position again, proving the elastic nature of the deformation. Additionally, a decrease in the band height ratio of the 1127 and 1097 cm(-1) bands was observed to go hand in hand with the straining of the fiber. This is assumed to reflect a widening of the torsion angle of the glycosidic C-O-C bonding. Thus, the 1097 cm(-1) band shift and the band height ratio enable one to follow the stretching of the cellulose at a molecular level, while the lignin bands are shown to be unaffected. Observed changes in the OH region are shown and interpreted as a weakening of the hydrogen-bonding network during straining. Future experiments on different native wood fibers with variable chemical composition and cellulose orientation and on chemically and enzymatically modified fibers will help to deepen the micromechanical understanding of plant cell walls and the associated macromolecules.     

Polarized vibrational spectroscopy of fiber polymers: hydrogen bonding in cellulose II

Vibrational spectroscopy using polarized incident radiation can be used to determine the orientation of X-H bonds with respect to coordinates such as crystallographic axes. The adaptation of this approach to polymer fibers is described here. It requires spectral intensity to be quantified around a 180 degrees range of polarization angles and not just recorded transversely and longitudinally as is normal in fiber spectroscopy. Mercerized cellulose II is used as an example. The unit cell of the cellulose II lattice contains six distinct hydroxyl groups engaged in a complex network of hydrogen bonds that hold the cellulose chains laterally together. A formalism is described to relate the variation in intensity of each O-H stretching mode to the angle between its transition moment and the chain axis as the polarization axis is rotated with respect to the fiber axis. It was necessary to include the effect of dispersion in chain orientation around the mean and the averaging of all rotational positions of the chains round their axis. The two crystallographically distinct O(2)-H groups, which are each hydrogen-bonded to only one acceptor oxygen, show a close match in orientation between the transition moments of their stretching bands and the O-H bond axis. The two O(3)-H groups each have a three-centered hydrogen bond to O-5 and O-6 of the next residue in the same chain. The transition moments of their stretching modes lay between the acceptor oxygens. Hydrogen bonding from the O(6)-H groups is still more complex but again the transition moment of each O-H bond lay within the cone of orientations described by the acceptor oxygens, provided that one additional acceptor oxygen excluded from the published crystal structure was considered. The transition moments for the O-H stretching modes were approximately aligned with the O-H bond axes, but the alignment was not necessarily exact. This approach is not restricted to hydroxyl groups, but it is particularly useful for the elucidation of hydrogen bonding in fibrous polymers for which crystallographic data on proton positions are not available.     

First-principles study of the structural transformation, electronic structure, and optical properties of crystalline 2,6-diamino-3,5-dinitropyrazine-1-oxide under high pressure

Periodic first-principles calculations have been performed to study the effect of high pressure on the geometric, electronic, and absorption properties of 2,6-diamino-3,5-dinitropyrazine-1-oxide (LLM-105) under hydrostatic pressures of 0–50 GPa. Obvious irregular changes in lattice constants, unit-cell angles, bond lengths, bond angles, and band gaps showed that crystalline LLM-105 undergoes four structural transformations at 8, 17, 25, and 42 GPa, respectively. The intramolecular H-bonds were strong at pressures of 0–41 GPa but weakened in the range 42–50 GPa. The lengths of the intermolecular H-bonds (<1.47 Å) indicated that these H-bonds have covalent character and tend to induce the formation of a new twelve-membered ring. Analysis of the DOS showed that the interactions between electrons, especially the valence electrons, strengthen under the influence of pressure. The p states play a very important role in chemical reactions of LLM-105. The absorption spectrum of LLM-105 displayed more bands—as well as stronger bands—in the fundamental absorption region when the pressure was high rather than low. A new absorption peak due to O–H stretching appeared at 18.3 eV above 40 GPa, indicating that covalent O–H bonds and a new twelve-membered ring are present in LLM-105.     

Sensing site‐specific structural characteristics and chirality using vibrational circular dichroism of isotope labeled peptides

Isotope labeling has a long history in chemistry as a tool for probing structure, offering enhanced sensitivity, or enabling site selection with a wide range of spectroscopic tools. Chirality sensitive methods such as electronic circular dichroism are global structural tools and have intrinsically low resolution. Consequently, they are generally insensitive to modifications to enhance site selectivity. The use of isotope labeling to modify vibrational spectra with unique resolvable frequency shifts can provide useful site‐specific sensitivity, and these methods have been recently more widely expanded in biopolymer studies. While the spectral shifts resulting from changes in isotopic mass can provide resolution of modes from specific parts of the molecule and can allow detection of local change in structure with perturbation, these shifts alone do not directly indicate structure or chirality. With vibrational circular dichroism (VCD), the shifted bands and their resultant sign patterns can be used to indicate local conformations in labeled biopolymers, particularly if multiple labels are used and if their coupling is theoretically modeled. This mini‐review discusses selected examples of the use of labeling specific amides in peptides to develop local structural insight with VCD spectra.     

Infrared spectra and hydrogen bonding of pentitols and pyranosides at 20 to 300 K

Infrared spectra in the range 400-4000 cm(-1) of three pentitols--ribitol, xylitol, D-arabinitol, and of three pyranosides--methyl alpha-D-manno-, methyl alpha-D-gluco- and methyl beta-D-galactopyranoside, as polycrystalline solids of both the pure OH and > 90% isotopically substituted OD compounds, were recorded at 20-300 K. In the low temperature spectra of the OH substances, at least three isolated narrow bands in the stretching mode and about ten narrow bands in the out-of-plane-bending mode range (< 1000 cm(-1)) are affected by cooling. Almost all have counterparts in the respective OD spectra with frequency ratios of 1.30-1.40. On this basis, they are assigned to OH groups bonded in H-bonds of different strengths (from 10 to 50 kJ mol(-1)). The average number of the OH...O hydrogen bonds is found to be two to three times larger than indicated by the stretching mode only or by structural data. The newly measured peak frequencies of the very narrow decoupled stretching mode bands show a correlation between the red shift (delta nu) and the H-bond length. As previously found for tetritols, the presence of weak H-bonds (bond energy < 14 kJ mol(-1)) is related to the different water sorption capabilities of the pentitols.     

Comparative structural analysis of cytidine, ethenocytidine and their protonated salts. II. IR spectral studies.

The IR spectra of crystalline cytidine (Cyd), ethenocytidine (epsilon Cyd), and their hydrochlorides (Cyd-Hcl and epsilon CyD-HCl) have been analyzed to determine the spectroscopic manifestations of the structural differences that were previously established for these nucleosides from X-ray studies. O,N-Deuteration of the samples turned out to be a successful approach to obtaining interpretable spectra. The analysis was carried out in three frequency ranges: (i) The 2600-1900 cm-1 range originating from the vO-D and VN-D vibrations. All intermolecular hydrogen bonds could be recognized here. The positions of the individual vO-D (vN-D) bands were correlated with the geometrical delta HB parameters presenting the strengths of hydrogen bonds in which these groups act as donors (ii) The 1750-1500 cm-1 region originating from the stretching vibrations of double bonds. All absorption bands in this region were interpreted in terms of electronic structures of the base fragments. (iii) The region of the C-H stretching vibrations of the base fragments (3200-3000 cm-1) and sugar moieties (3000-2800 cm-1). The Csp2-H vibrations also reflect the electronic structures of the base fragments, whereas the vCsp-H frequencies seem to be sensitive to etheno-bridging and to the presence of an intramolecular C6-H...05' hydrogen bond.     

Water structural changes in the bacteriorhodopsin photocycle: analysis by Fourier transform infrared spectroscopy.

The Fourier transform infrared difference spectra between light-adapted bacteriorhodopsin (BR) and its photointermediates, L and M, were analyzed for the 3750-3450-cm-1 region. The O-H stretching vibrational bands were identified from spectra upon substitution with 2H2O. Among them, the 3642-cm-1 band of BR was assigned to water by substitution with H2(18)O. By a comparison with the published infrared spectra of the water in model systems [Mohr, S.C., Wilk, W.D., & Barrow, G.M. (1965) J. Am. Chem. Soc. 87, 3048-3052], it is shown that the O-H bonds of the water in BR interact very weakly. Upon formation of L, the interaction becomes stronger. The O-H bonds of the protein side chain undergo similar changes. On the other hand, M formation further weakens the interaction of the same water molecules in BR. The appearance of a sharp band at 3486 cm-1, which was assigned tentatively to the N-H stretching vibration of the peptide bond, is unique to L. The results suggest that the water molecules are involved in the perturbation of Asp-96 in the L intermediate and that they are exerted from the protonated Schiff base which changes position upon the light-induced reaction.     

Stimulated Raman microscopy (CARS): From principles to applications

A new technique in microscopy is now available which permits to image specific molecular bonds of chemical species present in cells and tissues. The so called Coherent Anti-Stokes Raman Scattering (CARS) approach aims at maximizing the light matter interaction between two laser pulses and an intrinsic molecular vibrational level. This is possible through a non linear process which gives rise to a coherent radiation that is greatly enhanced when the frequency difference between the two laser pulses equals the Raman frequency of the aimed molecular bond. Similar to confocal microscopy, the technique permits to build an image of a molecular density within the sample but doesn't require any labelling or staining since the contrast uses the intrinsic vibrational levels present in the sample. Images of lipids in membranes and tissues have been reported together with their spectral analysis. In the case of very congested media, it is also possible to use a non invasive labelling such as deuterium which shifts the molecular vibration of the C-H bond down to the C-D bond range which falls in a silent region of the cell and tissue vibrational spectra. Such an approach has been used to study lipid phase in artificial membranes. Although the technique is still under development, CARS has now reach a maturity which will permit to bring the technology at a commercial stage in the near future. The last remaining bottleneck is the laser system which needs to be simplified but solutions are now under evaluation. When combined with others more conventional techniques, CARS should give its full potential in imaging unstained samples and like two photons techniques has the potential of performing deep tissues imaging.     

Effects of pH and chloride concentration on resonance Raman spectra of human myeloperoxidase and Raman microspectroscopic analysis of enzyme state in azurophilic granules

Resonance Raman spectra of human myeloperoxidase were examined at pH 3.3-10.5 in the absence and presence of chloride ions. Among the porphyrin vibrational bands, the core-size marker bands showed particularly large wavenumber downshifts on going from pH 8.7 to 5.3 with a transition midpoint at pH 6.5 in the absence of chloride ions. The chloride ions did not affect the spectrum at a pH below 5.3 and above 8.7 whereas an increase of chloride concentration at neutral pH caused spectral changes similar to those observed upon pH lowering. Analogous effects were also observed on the Raman intensity. In addition, the stretching mode of the bond between the heme Fe and proximal histidine shifted by -2 cm(-1) on going from pH 8.7 to 5.3. Decomposition of the nu(3) band revealed the presence of two components, which was confirmed by an isosbestic point in the absorption spectra. The observed spectral changes indicated the existence of alkaline and acidic forms of the enzyme. The pK of interconversion was 6.5, and it was increased by binding of chloride ions. The porphyrin core was slightly expanded in the acidic form compared to that in the alkaline form. A molecular mechanism of the porphyrin core expansion was proposed on the basis of the X-ray crystal structure. The pH-spectrum relationships obtained for the isolated enzyme were applied to in situ analysis of the state of myeloperoxidase in azurophilic granules of living neutrophils. The enzyme was stored in the acidic form and kept inactive in catalyzing HOCl production.     

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)