Resonance Raman Spectroscopic Data Research Articles

Insights into the charge-transfer character of electronic transitions in RCp2Ti(C2Fc)2 complexes using solvatochromism, resonance Raman spectroscopy, and TDDFT.

A series of complexes with low-energy FeII to TiIV metal-to-metal charge-transfer (MMCT) transitions, Cp2Ti(C2Fc)2, Cp*2Ti(C2Fc)2, and MeOOCCp2Ti(C2Fc)2, was investigated using solvatochromism and resonance Raman spectroscopy (RRS) augmented with time-dependent density functional theory (TDDFT) calculations in order to interrogate the nature of the CT transitions. Computational models were benchmarked against the experimental UV-Vis spectra and B3LYP/6-31G(d) was found to most faithfully represent the spectra. The energy of the MMCT transition was measured in 15 different solvents and a multivariate fit to the Catalán solvent parameters - solvent polarizability (SP), solvent dipolarity (SdP), solvent basicity (SB), and solvent acidity (SA) - was performed. The effect of SP indicates a greater degree of electron delocalization in the excited state (ES) than the ground state (GS). The small negative solvatochromism with respect to SdP indicates a smaller dipole moment in the ES than the GS. The effect of SB is consistent with charge-transfer to Ti. Upon excitation into the MMCT absorption band, the RRS data show enhancement of the alkyne stretching modes and of the out-of-plane bending modes of the cyclopentadienyl ring connected to Fe and the alkyne bridge. This is consistent with changes in the oxidation states of Ti and Fe, respectively. The higher-energy transitions (350-450 nm) show enhancement of vibrational modes consistent with ethnylcyclopentadienyl to Ti ligand-to-metal charge transfer (LMCT). The RRS data is consistent with the TDDFT predicted character of these transitions. TDDFT suggests that the lowest-energy transition in Cp2Ti(C2Fc)2CuI, where CuI is coordinated between the alkynes, retains its FeII to TiIV MMCT character, in agreement with the RRS data, but that the lowest-energy transitions have significant CuI to Ti character. For Cp2Ti(C2Fc)2CuI, excitation into the low-energy MMCT absorption band results in selective enhancement of the symmetric alkynyl stretching mode.

Photodissociation of Protonated Species of Amino- and Dimethylaminoazobenzene, According to Resonance Raman Spectroscopy Data

Literature data on the resonance Raman spectroscopy of amino- and dimethylaminoazobenzene are analyzed based on concepts formulated earlier for the rydimeric structure of amino azo dyes. The traditional monomeric–quinoid theory of tautomerism and color is shown to be inadequate when attributing bands in the Raman spectra of protonated species of amino- and dimethylaminoazobenzene. Resonance Raman spectra are adequately characterized using the concept of the rydimeric structure of protonated species. It is revealed that protonated rydimers become strong acids upon photoexcitation.

Probing Framework-Restricted Metal Axial Ligation and Spin State Patterns in a Post-Synthetically Reduced Iron-Porphyrin-Based Metal-Organic Framework.

An iron-porphyrin-based metal organic framework PCN-222(Fe) is investigated upon postsynthetic reduction with piperidine. Fe K-edge X-ray absorption and Kβ mainline emission spectroscopy measurements reveal the local coordination geometry, oxidation, and spin state changes experienced by the Fe sites upon reaction with this axially coordinating reducing agent. Analysis and fitting of these data confirm the binding pattern predicted by a space-filling model of the structurally constrained pore environments. These results are further supported by UV-vis diffuse reflectance, IR, and resonance Raman spectroscopy data.

Transmembrane Substrate Unfolding in Intramembrane Proteolysis

In recent years impressive progress has been made in our understanding of intramembrane cleaving proteases (iCliPs), enzymes that carry out proteolytic reactions within cell membranes, and their substrates, but basic biochemical questions such as what determines a transmembrane helix to be a substrate and how cleavage site is dictated remain unresolved. Much onus as to why we sill do not understand substrate specificity in these enzymes falls to the difficulties in monitoring enzyme-substrate interactions in a lipid or membrane-mimetic environment. Here we will present deep-UV resonance Raman spectroscopy data describing discrete structural characteristics of substrates prior to and during interactions with iCLiPS indicating structural flexibility in both environments is an intrinsic property of cleavable substrates. Furthermore, we also will present evidence that mutagenesis of substrates that abolishes intramembrane proteolysis do not significantly influence binding and dissociation constants of enzyme and substrate, while they do convey distinct changes to the intrinsic structural flexibility of the substrate. The mutations also alter the extent to which the enzyme influences the substrate structure during binding. Overall these results imply a synergy between a sequence complementarity between enzyme and substrate, which requires adequate structural flexibility of the substrate transmembrane helical sequence in order to expose the peptide bond for cleavage.

How Does Peripheral Functionalization of Ruthenium(II)-Terpyridine Complexes Affect Spatial Charge Redistribution after Photoexcitation at the Franck-Condon Point?

Ruthenium polypyridine-type complexes are extensively used sensitizers to convert solar energy into chemical and/or electrical energy, and they can be tailored through their metal-to-ligand charge-transfer (MLCT) properties. Much work has been directed at harnessing the triplet MLCT state in photoinduced processes, from sophisticated molecular architectures to dye-sensitized solar cells. In dye-sensitized solar cells, strong coupling to the semiconductor exploits the high reactivity of the (hot) singlet/triplet MLCT state. In this work, we explore the nature of the (1) MLCT states of remotely substituted Ru(II) model complexes by both experimental and theoretical techniques. Two model complexes with electron-withdrawing (i.e. NO2 ) and electron-donating (i.e. NH2 ) groups were synthesized; these complexes contained a phenylene spacer to serve as a spectroscopic handle and to confirm the contribution of the remote substituent to the (1) MLCT transition. [Ru(tpy)2 ](2+) -based complexes (tpy=2,2':6',2''-terpyridine) were further desymmetrized by tert-butyl groups to yield unidirectional (1) MLCTs with large transition dipole moments, which are beneficial for related directional charge-transfer processes. Detailed comparison of experimental spectra (deconvoluted UV/Vis and resonance Raman spectroscopy data) with theoretical calculations based on density functional theory (including vibronic broadening) revealed different properties of the optically active bright (1) MLCT states already at the Franck-Condon point.

Raman spectroscopy for probing covalent functionalization of single-wall carbon nanotubes bundles with gold nanoparticles

A hybrid system made of single-wall carbon nanotube bundles (average diameter of approximately 20 nm and length of several tens of nanometers) highly covered with gold nanoparticles (average diameter of 5 nm) was prepared through the functionalization of the nanotube surface with 4,4′-thiobisbenzenethiol molecules followed by the anchoring of gold nanoparticles. The decoration of single wall carbon nanotubes with gold nanoparticles was performed using two different methods, named as ex situ and in situ, which refer to the gold reduction before or during the contact with the nanotubes, respectively. Transmission electron microscopy images showed that both methods lead to a successful decoration of the single wall carbon nanotube bundles, although different density of gold nanoparticles covering the bundles was observed to depend on the reaction methods. Resonance Raman spectroscopy data were used to follow the electronic changes of the carbon nanotubes after gold nanoparticles loading and confirmed the strong interactions of the gold nanoparticles with the 4,4′-thiobisbenzenethiol molecules and the carbon nanotubes. This interaction was probed in the Raman spectrum which unveiled a surface enhancement Raman effect of the thiol molecule Raman signals, not observed before the attachment of metallic nanoparticles onto 4,4′-thiobisbenzenethiol functionalized carbon nanotubes.

Precursor Transformation during Molecular Oxidation Catalysis with Organometallic Iridium Complexes

We present evidence for Cp* being a sacrificial placeholder ligand in the [Cp*Ir(III)(chelate)X] series of homogeneous oxidation catalysts. UV-vis and (1)H NMR profiles as well as MALDI-MS data show a rapid and irreversible loss of the Cp* ligand under reaction conditions, which likely proceeds through an intramolecular inner-sphere oxidation pathway reminiscent of the reductive in situ elimination of diolefin placeholder ligands in hydrogenation catalysis by [(diene)M(I)(L,L')](+) (M = Rh and Ir) precursors. When oxidatively stable chelate ligands are bound to the iridium in addition to the Cp*, the oxidized precursors yield homogeneous solutions with a characteristic blue color that remain active in both water- and CH-oxidation catalysis without further induction period. Electrophoresis suggests the presence of well-defined Ir-cations, and TEM-EDX, XPS, (17)O NMR, and resonance-Raman spectroscopy data are most consistent with the molecular identity of the blue species to be a bis-μ-oxo di-iridium(IV) coordination compound with two waters and one chelate ligand bound to each metal. DFT calculations give insight into the electronic structure of this catalyst resting state, and time-dependent simulations agree with the assignments of the experimental spectroscopic data. [(cod)Ir(I)(chelate)] precursors bearing the same chelate ligands are shown to be equally effective precatalysts for both water- and CH-oxidations using NaIO4 as chemical oxidant.

Site-specific covalent attachment of heme proteins on self-assembled monolayers

Naturally occurring hemin cofactor has been functionalized to introduce two terminal alkyne groups. This modified hemin has been successfully covalently attached to mixed self-assembled monolayers of alkanethiols and azide-terminated alkanethiols on gold electrodes using a Cu(I)-catalyzed 1,3-cycloaddition reaction. However these hemin-modified electrodes could not be used to reconstitute apomyoglobin on gold electrodes owing to the hydrophobicity of the alkane thiol self-assembled monolayer. Modification of existing techniques allowed covalent attachment of alkyne-terminated electroactive species onto mixed monolayers of azidothiols and carboxylatoalkanethiols on electrodes using the same Cu(I)-catalyzed 1,3-cycloaddition reaction. Apomyoglobin could be reconstituted using the hemin covalently attached to these hydrophilic electrodes. The electrochemical data, UV-vis absorption data, surface-enhanced resonance Raman spectroscopy data, and atomic force microscopy data indicate the presence of these modified myoglobin proteins on these electrodes. The direct attachment of the heme cofactor of these modified myoglobin proteins to the electrode allows fast electron transfer to the heme center from the electrode and affords efficient O(2)-reducing bioelectrodes under physiological conditions.

Sulfide Binding Properties of Truncated Hemoglobins

The truncated hemoglobins from Bacillus subtilis (Bs-trHb) and Thermobifida fusca (Tf-trHb) have been shown to form high-affinity complexes with hydrogen sulfide in their ferric state. The recombinant proteins, as extracted from Escherichia coli cells after overexpression, are indeed partially saturated with sulfide, and even highly purified samples still contain a small but significant amount of iron-bound sulfide. Thus, a complete thermodynamic and kinetic study has been undertaken by means of equilibrium and kinetic displacement experiments to assess the relevant sulfide binding parameters. The body of experimental data indicates that both proteins possess a high affinity for hydrogen sulfide (K = 5.0 x 10(6) and 2.8 x 10(6) M(-1) for Bs-trHb and Tf-trHb, respectively, at pH 7.0), though lower with respect to that reported previously for the sulfide avid Lucina pectinata I hemoglobins (2.9 x 10(8) M(-1)). From the kinetic point of view, the overall high affinity resides in the slow rate of sulfide release, attributed to hydrogen bonding stabilization of the bound ligand by distal residue WG8. A set of point mutants in which these residues have been replaced with Phe indicates that the WG8 residue represents the major kinetic barrier to the escape of the bound sulfide species. Accordingly, classical molecular dynamics simulations of SH(-)-bound ferric Tf-trHb show that WG8 plays a key role in the stabilization of coordinated SH(-) whereas the YCD1 and YB10 contributions are negligible. Interestingly, the triple Tf-trHb mutant bearing only Phe residues in the relevant B10, G8, and CD1 positions is endowed with a higher overall affinity for sulfide characterized by a very fast second-order rate constant and 2 order of magnitude faster kinetics of sulfide release with respect to the wild-type protein. Resonance Raman spectroscopy data indicate that the sulfide adducts are typical of a ferric iron low-spin derivative. In analogy with other low-spin ferric sulfide adducts, the strong band at 375 cm(-1) is tentatively assigned to a Fe-S stretching band. The high affinity for hydrogen sulfide is thought to have a possible physiological significance as H(2)S is produced in bacteria at metabolic steps involved in cysteine biosynthesis and hence in thiol redox homeostasis.

A resonance Raman spectroscopic investigation into the effects of fixation and dehydration on heme environment of hemoglobin

AbstractThe effects of fixation and dehydration procedures on heme environment inside human erythrocytes were examined using resonance Raman spectroscopy. The resonance Raman spectroscopic data along with far‐infrared spectra show that hemoglobin in air‐dried red blood cells and those fixed in the precipitating fixatives, methanol and acetone, changes to hemichrome, a low‐spin component in which the sixth coordination site of the iron is occupied by the imidazole group of the distal histidine. The cross‐linking fixatives, formaldehyde and glutaraldehyde, maintain hemoglobin in its native conformational state (oxyhemoglobin) as evidenced by the appearance of the oxygen ligand marker bands along with the skeletal sensitive modes. However, polymerization with cross‐linking fixatives increases the auto‐oxidation kinetics of hemoglobin and gradually oxidizes it to the met state. Moreover, the dehydration of a red blood cell under N2 results in a change of oxyhemoglobin to a six coordinate low‐spin FeII derivative of hemoglobin, hemochrome. Copyright © 2009 John Wiley & Sons, Ltd.

Selective Tuning of the Electronic Properties of Coaxial Nanocables through Exohedral Doping

The electronic properties of exohedrally doped double-walled carbon nanotubes (DWNTs) have been investigated using density functional theory and resonance Raman spectroscopy (RRS) measurements. First-principles calculations elucidate the effects of exohedral doping on the M@S and S@M systems, where a metallic (M) tube is either inside or outside a semiconducting (S) one. The results demonstrate that metallic nanotubes are extremely sensitive to doping even when they are inner tubes, in sharp contrast to semiconducting nanotubes, which are not affected by doping when the outer shell is a metallic nanotube (screening effects). The theoretical predictions are in agreement with RRS data on Br2- and H2SO4-doped DWNTs. These results pave the way to novel nanoscale electronics via exohedral doping.

Electronic characterization and photocatalytic properties of CdS/TiO 2 semiconductor composite

Titanium dioxide is broadly used as a catalyst for photochemical reactions. In this work, nanometric particles of CdS (a narrow band gap semiconducting material) were used to impregnate TiO 2 in order to optimize its photocatalytic properties. CdS/TiO 2 semiconductor composites were obtained in 1, 3, 5 and 20 mol% proportion and characterized by differential thermogravimetric analysis (DTG), X-ray diffraction (XRD), laser-induced fluorescence (LIF) and resonance Raman spectroscopy. The results identified the CdS in hexagonal geometry and the TiO 2 in anatase form. Resonance Raman spectroscopy data revealed a lattice softening of the TiO 2-anatase phase due to electronic interaction between TiO 2 and CdS in the CdS/TiO 2 catalyst. The effect of modifying the proportion of CdS in the catalyst electronic properties and efficiency was also studied. The photocatalytic activity of the CdS/TiO 2 was investigated using artificial UV light for degradation of the textile azo-dye Drimaren red. The photocatalytic analysis revealed better efficiency for the CdS/TiO 2 5% composite as compared with other CdS/TiO 2 proportions and with TiO 2 alone.

A conformational mimic of the MgATP-bound "on state" of the nitrogenase iron protein.

The crystal structure of a nitrogenase Fe protein single site deletion variant reveals a distinctly new conformation of the Fe protein and indicates that, upon binding of MgATP, the Fe protein undergoes a dramatic conformational change that is largely manifested in the rigid-body reorientation of the homodimeric Fe protein subunits with respect to one another. The observed conformational state allows the rationalization of a model of structurally and chemically complementary interactions that occur upon initial complex formation with the MoFe protein component that are distinct from the protein-protein interactions that have been characterized previously for stabilized nitrogenase complexes. The crystallographic results, in combination with complementary UV-visible absorption, EPR, and resonance Raman spectroscopic data, indicate that the [4Fe-4S] cluster of both the Fe protein deletion variant and the native Fe protein in the presence of MgATP can reversibly cycle between a regular cubane-type [4Fe-4S] cluster in the reduced state and a cleaved form involving two [2Fe-2S] fragments in the oxidized state. Resonance Raman studies indicate that this novel cluster conversion is induced by glycerol, and the crystallographic data suggest that glycerol is bound as a bridging bidentate ligand to both [2Fe-2S] cluster fragments in the oxidized state.

Three-coordinate copper(II)-phenolate complexes.

The reactions of LCuCl (L = 2,4-bis((2,6-diisopropylphenyl)imido)pentane (L(iPr)), 2,4-bis((2,6-diisopropylphenyl)imido)-3-chloropentane (L(CliPr))) with the phenolates TlOAr (Ar = C(6)H(3)Me(2), C(6)H(4)OMe, C(6)H(4)tBu) and NaOC(6)H(3)(tBu)(2) were explored. Novel three-coordinate Cu(II)-phenolates, LCuOAr, were isolated from the reactions with the thallium phenolates and were characterized by X-ray crystallography and spectroscopy (UV-vis, EPR). The complexes feature short Cu-O(phenolate) distances (average Cu-O = 1.81 A) and, with one exception, irregular N-Cu-O(phenolate) angles that differ within each compound (15 degrees < Delta < 28 degrees, where Delta = angleN(1)-Cu-O - angleN(2)-Cu-O). The exception is L(iPr)Cu(OC(6)H(4)tBu), for which X-ray structures at -100 and 25 degrees C differed due to an unusual reversible phase change with nonmerohedral twinning (2:1 ratio) in the low-temperature form. The high-temperature form has local C(2)(v) symmetry (Delta = 0 degrees ), and upon cooling below the phase transition temperature (-8 +/- 5 degrees C) lateral movement of the phenolate ligand (Delta = 17.6 degrees ) and rotation of the phenolate plane by 10.7 degrees occurs. Resonance Raman spectroscopic data acquired for L(iPr)Cu(OC(6)H(4)tBu) corroborated assignment of phenolate --> Cu(II) LMCT character in the UV-vis spectra. Cyclic voltammetry experiments (THF, 0.5 M NBu(4)PF(6)) revealed negative E(1/2) values for the Cu(II)/Cu(I) couples relative to NHE, consistent with enhanced stabilization of the Cu(II) state by both the strongly electron donating beta-diketiminate ligand and the phenolates. Although thermally stable, the Cu(II)-phenolates are unusually reactive with dioxygen, albeit to give product(s) that have yet to be identified. In the reaction of L(iPr)CuCl with NaOC(6)H(3)(tBu)(2) no Cu(II)-phenolate was observed. Instead, a Cu(I) complex was generated quantitatively by trapping with added isocyanide, [L(iPr)CuNC(C(6)H(3)Me(2))], along with 3,3',5,5'-tetra-tert-butyl-4,4'-dibenzoquinone and 2,6-di-tert-butylphenol in 27 +/- 3% and 46 +/- 6% yields, respectively, corresponding to the overall reaction 4L(iPr)Cu(II)Cl + 4NaOAr --> 4L(iPr)Cu(I) + 4NaCl + dibenzoquinone + 2(phenol).

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.

Characterization of a Diiron(III) Peroxide Intermediate in the Reaction Cycle of Methane Monooxygenase Hydroxylase from Methylococcus capsulatus (Bath)

We present new optical and resonance Raman spectroscopic data that characterize the first intermediate as a diiron(III) peroxo species. The time-resolved optical and freeze-quench resonance Raman spectroscopic experiments strongly support assignment of the first intermediate, variously designated P or L, in the MMO hydroxylase reaction cycle as a symmetrical diiron(III) peroxo species. 32 refs., 2 figs.

Vibrational mode analysis of 2-aminoadenine and its deuterated species from Raman and ultraviolet resonance Raman data

Resonance Raman spectroscopy data of 2-aminoadenine and its deuterated species (C8-deuterated, N-deuterated and C8-, N-deuterated derivatives) in aqueous solution have been collected in the spectral region between 400 and 1800 cm−1, by using ultraviolet excitation wavelengths (λexc = 222, 257 and 281 nm) located in the three main UV absorption bands corresponding to the strongly allowed electronic transitions of the molecule of interest. Moreover, a Raman spectrum has been recorded under off-resonance conditions with a visible excitation (λexc= 488 nm). In order to assign the 2-aminoadenine in-plane vibrational bands displayed in the RRS spectra, a normal coordinate analysis has been performed by means of an empirical internal valence force field. These calculations are based on our recent normal mode analysis of adenine and guanine nucleic bases and their deuterated species, which was based on the joint use of resonance Raman spectroscopy and neutron inelastic scattering data. In the 2-aminoadenine force field proposed here, the diagonal force constants have been directly transferred from those recently obtained for adenine (and from guanine as concerns the 2-amino group), the interaction force constants (off-diagonal) then being adjusted on the basis of the actual experimental data from 2-aminoadenine and its deuterated species. The current force field is also able to assign infrared and Raman data obtained by other authors from polycrystalline samples of the pure species.

Protein: nucleic acid interactions. I. Electronic structures of cytosine, indole, and guanine complexes.

Low singlet transition energies and line strengths were calculated for the cytosine:indole:guanine complex by the INDO/1S-CI method. The chromophores were arranged in three sets of 270 intercalating geometries. Calculations were executed in the supermolecule model with single excited configurations. Errors due to basis set extension and incomplete configuration representation were assessed, for all chromophore pairs, by full BSSE correction calculations and inclusion of double-excited configurations. The intercalation-induced perturbations of the principal transitions are characterized by but not limited to (a) a decrease in strength of [pi*,pi] transitions, (b) increase in strength in [pi*,n] transitions, (c) splitting of [pi*,pi] transitions into components of unequal strength, and (d) energy and strength dependence in mixed transitions on rise and shift movements of the nucleic acid bases. These predictions are in accord with absorption, fluorescence emission, and scattering, and resonance Raman spectroscopic data on oligonucleotides and analogous aromatic complexes. The calculations suggest that major differences in intercalating coordinations are discernible in the near-uv spectroscopic domain of proteins and nucleic acids.

A quantum chemical study of the degradation and the maximum polyene length in PVC

The semi-empirical MINDO/3, AM1 and MNDO-PM3 methods were employed to study the stability of polyenes, polyenyl cations and polyenyl radicals. Both cations and radicals were subjected to computation since the elimination of HCl from PVC might proceed according to an ionic or radical mechanism. We subsequently investigated the effect of additional alkane chain ends, as well as PVC-like chain ends to both ends of the polyene sequence, in order to mimic degraded PVC better. The results are discussed in relation to the experimentally observed polyene length distribution in degraded PVC, as revealed from UV-Vis and Resonance Raman Spectroscopic data. The following conclusions may be drawn from the data presented. The MINDO/3 method has to be rejected. From the calculations, which basically refer to the molecules being in the gas phase, it was concluded that the formation of radical intermediates during polyene formation is much preferred (energetically) as compared to the corresponding ionic species. However, several researchers have suggested that the ionic process is preferred in solid PVC (‘solvation effect’); for solid PVC hard experimental evidence is still lacking with respect to either of the two mechanisms. The introduction of saturated chain ends markedly influences the energetics and the stability of the polyenes. HCl abstraction from an intact PVC chain was calculated to be about as probable as HCl abstraction adjacent to a polyene sequence. Calculations on polyenes with PVC-like chain ends do not give any indication that there is a maximum polyene length after which further elimination of HCl becomes less probable from an energetic point of view. The calculated data presented show no evidence for a finite polyene length, and thus do not support the unzipping mechanism. We propose a new one-step unzipping mechanism.

Identity of acyl group conformations in the active sites of papain and cathepsin B by resonance Raman spectroscopy.

Resonance Raman spectroscopic data provide conclusive evidence for the existence of an acyl-enzyme intermediate during the reaction of a thionoester substrate, N-methyloxycarbonylphenylalanylglycine methyl thionoester (CH3OC(=O)-Phe-NHCH2C(=S) OCH3), with cathepsin B from porcine spleen. The resonance Raman spectrum of CH3OC(=O)-Phe-NHCH2C(=S)S-cathepsin B, where the thiol S is from the active-site cysteine residue, is compared to that of the corresponding papain acyl-enzyme. Within the limits of experimental error (+/-2 cm-1 for peak positions), there are no detectable spectral differences. Since the resonance Raman spectrum is sensitive to the torsional angles in the glycinic bonds and the cysteine linkages, the conformations are identical in those parts of the acyl-enzymes where chemical transformation occurs. A conformational analysis of the model compound CH3OC(=O)-Phe-NHCH2C(=S)SC2H5 demonstrates that the dithioacyl group in both dithioacyl-enzymes is present as a single population of a form known as conformer B. Conformer B is characterized by a small torsional angle about the glycinic NHCH2-CS(thiol) bond such that the nitrogen and S (thiol) atoms are in close contact. This conformer is widespread among the dithioacyl intermediates of plant cysteine proteinases, and it is apparent that the same chemistry is retained in a mammalian cysteine proteinase. Steady-state kinetic parameters are also reported for CH3OC(=O)-Phe-NHCH2C(=S)OCH3 reacting with papain and cathepsin B. The similarity of the Kcat values, 0.53 and 1.15 s-1, for papain and cathepsin B, respectively, provides further evidence for a conserved deacylation process.