Spectroscopic studies of the anaerobic enzyme-substrate complex of catechol 1,2-dioxygenase

The basis of the respective regiospecificities of intradiol and extradiol dioxygenase is poorly understood and may be linked to the protonation state of the bidentate-bound catechol in the enzyme/substrate complex. Previous ultraviolet resonance Raman (UVRR) and UV-visible (UV-vis) difference spectroscopic studies demonstrated that, in extradiol dioxygenases, the catechol is bound to the Fe(II) as a monoanion. In this study, we use the same approaches to demonstrate that, in catechol 1,2-dioxygenase (C12O), an intradiol enzyme, the catechol binds to the Fe(III) as a dianion. Specifically, features at 290 nm and 1550 cm(-1) in the UV-vis and UVRR difference spectra, respectively, are assigned to dianionic catechol based on spectra of the model compound, ferric tris(catecholate). The UVRR spectroscopic band assignments are corroborated by density functional theory (DFT) calculations. In addition, negative features at 240 nm in UV-vis difference spectra and at 1600, 1210, and 1175 cm(-1) in UVRR difference spectra match those of a tyrosinate model compound, consistent with protonation of the axial tyrosinate ligand when it is displaced from the ferric ion coordination sphere upon substrate binding. The DFT calculations ascribe the asymmetry of the bound dianionic substrate to the trans donor effect of an equatorially ligated tyrosinate ligand. In addition, the computations suggest that trans donation from the tyrosinate ligand may facilitate charge transfer from the substrate to yield the iron-bound semiquinone transition state, which is capable of reacting with dioxygen. In illustrating the importance of ligand trans effects in a biological system, the current study demonstrates the power of combining difference UVRR and optical spectroscopies to probe metal ligation in solution.     

UV resonance raman investigation of electronic transitions in alpha-helical and polyproline II-like conformations

UV resonance Raman (UVRR) excitation profiles and Raman depolarization ratios were measured for a 21-residue predominantly alanine peptide, AAAAA(AAARA) 3A (AP), excited between 194 and 218 nm. Excitation within the pi-->pi* electronic transitions of the amide group results in UVRR spectra dominated by amide vibrations. The Raman cross sections and excitation profiles provide information about the nature of the electronic transitions of the alpha-helix and polyproline II (PPII)-like peptide conformations. AP is known to be predominantly alpha-helical at low temperatures and to take on a PPII helix-like conformation at high temperatures. The PPII-like and alpha-helix conformations show distinctly different Raman excitation profiles. The PPII-like conformation cross sections are approximately twice those of the alpha-helix. This is due to hypochromism that results from excitonic interactions between the NV 1 transition of one amide group with higher energy electronic transitions of other amide groups, which decreases the alpha-helical NV 1 (pi-->pi*) oscillator strengths. Excitation profiles of the alpha-helix and PPII-like conformations indicate that the highest signal-to-noise Raman spectra of alpha-helix and PPII-like conformations are obtained at excitation wavelengths of 194 and 198 nm, respectively. We also see evidence of at least two electronic transitions underlying the Raman excitation profiles of both the alpha-helical and the PPII-like conformations. In addition to the well-known approximately 190 nm pi-->pi* transitions, the Raman excitation profiles and Raman depolarization ratio measurements show features between 205-207 nm, which in the alpha-helix likely results from the parallel excitonic component. The PPII-like helix appears to also undergo excitonic splitting of its pi-->pi* transition which leads to a 207 nm feature.     

A UV resonance Raman (UVRR) spectroscopic study on the extractable compounds in Scots pine (Pinus sylvestris) wood. Part II. Hydrophilic compounds

Hydrophilic extracts of Scots pine (Pinus sylvestris) heartwood and sapwood and a solid Scots pine knotwood sample were studied by UV resonance Raman spectroscopy (UVRRS). In addition, UVRR spectra of two hydrophilic model compounds (pinosylvin and chrysin) were analysed. UV Raman spectra were collected using 244 and 257 nm excitation wavelengths. The chemical composition of the acetone:water (95:5 v/v) extracts were also determined by gas chromatography. The aromatic and oleophilic structures of pinosylvin and chrysin showed three intense resonance enhanced bands in the spectral region of 1649-1548 cm(-1). Pinosylvin showed also a relatively intense band in the aromatic substitution region at 996 cm(-1). The spectra of the heartwood acetone:water extract showed many bands typical of pinosylvin. In addition, the extract included bands distinctive for resin and fatty acids. The sapwood acetone:water extract showed bands due to oleophilic structures at 1655-1650 cm(-1). The extract probably also contained oligomeric lignans because the UVRR spectra were in parts similar to that of guaiacyl lignin. The characteristic band of pinosylvin (996 cm(-1)) was detected in the UVRR spectrum of the resin rich knotwood. In addition, several other bands typical for wood resin were observed, which indicated that the wood resin in the knotwood was resonance enhanced even more than lignin.     

Direct detection of 8-oxo-deoxyguanosine using UV resonance Raman spectroscopy

8-oxo-deoxyguanosine (8-oxo-dG) is a major oxidative lesion in DNA and is responsible for mutation and cancer. Current techniques for detecting 8-oxo-dG are indirect methods. Thus, development of new methodologies is needed to directly detect such oxidative lesions. In this article, we have used ultraviolet resonance Raman (UVRR) spectroscopy as a novel analytical technique for the detection of 8-oxo-dG. Here, the UVRR spectrum of 8-oxo-dG was acquired and compared to that of deoxyguanosine (dG) and deoxyadenosine (dA). Data analysis shows a distinct UVRR spectrum of 8-oxo-dG with characteristic peaks. Detection of 8-oxo-dG was easily achieved from a mixture with dG. These results reveal that UVRR spectroscopy shows promise as a direct method for detecting 8-oxo-dG.     

The near ultraviolet absorption spectra of 2,3-, 2,4- and 2,5-dimethylbenzonitriles

A new technique—photoacoustic spectroscopy (PAS—was applied for the study of the electronic transitions in three isomers, 2,3-, 2,4- and 2,5-dimethylbenzonitriles. The PAS spectra were compared with solution and vapour phase spectra. A probable detection of S → T absorption in the molecules is considered to be of significance in the present investigation. It is also shown that two π → π* transitions analogous to the benzene strong 200 nm and weak and forbidden 260 nm transitions could be identified in all three molecules. An interesting feature is regarding the identification of a few combination bands in the excited state of an electronic transition which could be fairly comparable with such combinations observed in the near i.r. PAS spectra of the molecules, in the ground state.     

Initial excited-state structural dynamics of 2'-deoxyguanosine determined via UV resonance Raman spectroscopy

The resonance Raman spectra of 2'-deoxyguanosine, a DNA nucleoside, were measured in aqueous solution at wavelengths throughout its 260 nm absorption band. Self-consistent analysis of the resulting resonance Raman excitation profiles and absorption spectrum using a time-dependent wave packet formalism with two electronic states yielded the initial excited-state structural dynamics in both states. The vibrational modes containing the N(7)═C(8) stretching and C(8)-H bending internal coordinates were found to exhibit significant initial structural dynamics upon photoexcitation to either state and are coincident with the photochemical reaction coordinate involving the formation of the 2'-deoxyguanosine cation radical.     

Tunable resonance hyper-Raman spectroscopy of second-order nonlinear optical chromophores

Two-photon-resonant hyper-Raman spectra are reported for three "push-pull" conjugated organic chromophores bearing -NO(2) acceptor groups, two dipolar and one octupolar. The excitation source is an unamplified picosecond mode-locked Ti:sapphire laser tunable from 720 to 950 nm. The linear resonance Raman spectra of the same molecules are measured using excitation from the laser second harmonic. Excitation on resonance with the lowest-lying band in the linear absorption spectrum yields nearly identical resonance Raman and resonance hyper-Raman spectra. However, excitation into a region that appears to contain more than one electronic transition gives rise to different intensity patterns in the linear and nonlinear spectra, indicating that different transitions contribute differently to the one-photon and two-photon oscillator strength. The promise of the hyper-Raman technique for examining electronic transitions that are both one- and two-photon allowed is discussed.     

Density functional theory and Raman spectroscopy applied to structure and vibrational mode analysis of 1,1',3,3'-tetraethyl-5,5',6,6'-tetrachloro- benzimidazolocarbocyanine iodide and its aggregate

We have measured electronic and Raman scattering spectra of 1,1',3,3'-tetraethyl-5,5',6,6'-tetrachloro-benzimidazolocarbocyanine iodide (TTBC) in various environments, and we have calculated the ground state geometric and spectroscopic properties of the TTBC cation in the gas and solution phases (e.g., bond distances, bond angles, charge distributions, and Raman vibrational frequencies) using density functional theory. Our structure calculations have shown that the ground state equilibrium structure of a cis-conformer lies ～200 cm(-1) above that of a trans-conformer and both conformers have C(2) symmetry. Calculated electronic transitions indicate that the difference between the first transitions of the two conformers is about 130 cm(-1). Raman spectral assignments of monomeric- and aggregated-TTBC cations have been aided by density functional calculations at the same level of the theory. Vibrational mode analyses of the calculated Raman spectra reveal that the observed Raman bands above 700 cm(-1) are mainly associated with the in-plane deformation of the benzimidazolo moieties, while bands below 700 cm(-1) are associated with out-of-plane deformations of the benzimidazolo moieties. We have also found that for the nonresonance excited experimental Raman spectrum of aggregated-TTBC cation, the Raman bands in the higher-frequency region are enhanced compared with those in the nonresonance spectrum of the monomeric cation. For the experimental Raman spectrum of the aggregate under resonance excitation, however, we find new Raman features below 600 cm(-1), in addition to a significantly enhanced Raman peak at 671 cm(-1) that are associated with out-of-plane distortions. Also, time-dependent density functional theory calculations suggest that the experimentally observed electronic transition at ～515 nm (i.e., 2.41 eV) in the absorption spectrum of the monomeric-TTBC cation predominantly results from the π → π? transition. Calculations are further interpreted as indicating that the observed shoulder in the absorption spectrum of TTBC in methanol at 494 nm (i.e., 2.51 eV) likely results from the ν(") = 0 → ν' = 1 transition and is not due to another electronic transition of the trans-conformer-despite the fact that measured and calculated NMR results (not provided here) support the prospect that the shoulder might be attributable to the 0-0 band of the cis-conformer.     

Structural changes upon reduction of dipyrido[2,3-a:3',2'-c]phenazine probed by vibrational spectroscopy, ab initio calculations, and deuteration studies

A series of bridging ligands, dipyrido[2,3-a:3',2'-c]phenazine (ppb), dipyrido[2,3-a:3',2'-c]-6,7-dichlorophenazine (ppbCl2), and dipyrido[2,3-a:3',2'-c]-6,7-dimethylphenazine (ppbMe2), and their binuclear copper(I) complexes have been synthesized, and their spectral properties were measured. The single-crystal structure of the complex, [(PPh3)2Cu(mu-ppbCl2)Cu(PPh3)2](BF4)2 in the monoclinic space group P21/c, 18.2590(1), 21.1833(3), 23.2960(3) A with Z = 4 is reported. The copper(I) complexes are deeply colored through MLCT transitions in the visible region. The vibrational spectra of the ligands have been modeled using ab initio hybrid density functional theory (DFT) methods (B3LYP/6-31G(d)) and compared to experimental FT-Raman and IR data. The DFT calculations are used to interpret the resonance Raman spectra, and thus the electronic spectra, of the complexes. The preferential enhancement of modes associated with the phenanthroline section of the ligands with blue excitation (lambda(exc) = 457.9 nm) over phenazine-based modes with redder excitation (lambda(exc) = 514.5 and 632.8 nm) suggests the 2 MLCT transitions terminated on different unoccupied MOs are present under the visible absorption envelope. The radical anion species of the ligands are prepared by the electrochemical reduction of the binuclear copper(I) complexes; no evidence of dechelation prevalent in other copper(I) complexes is observed. The resonance Raman spectra of the reduced complexes are dramatically different from those of the parent species. Across the series common bands are observed at about 1590 and 1570 cm(-1) which do not shift with reduction but are altered in intensity. The normal-mode analysis of the radical anion species suggests that these normal modes primarily involve bond length distortions that are unaffected by reduction.     

Resonance hyper-Raman excitation profiles and two-photon states of a donor-acceptor substituted polyene

Resonance Raman and resonance hyper-Raman spectra and excitation profiles have been measured for a "push-pull" donor-acceptor substituted conjugated polyene bearing a julolidine donor group and a nitrophenyl acceptor group, in acetone at excitation wavelengths from 485 to 356 nm (two-photon wavelengths for the nonlinear spectra). These wavelengths span the strong visible to near-UV linear absorption spectrum, which appears to involve at least three different electronic transitions. The relative intensities of different vibrational bands vary considerably across the excitation spectrum, with the hyper-Raman spectra showing greater variation than the linear Raman. A previously derived theory of resonance hyper-Raman intensities is modified to include contributions from purely vibrational levels of the ground electronic state as intermediate states in the two-photon absorption process. These contributions are found to have only a slight effect on the hyper-Rayleigh intensities and profiles, but they significantly influence some of the hyper-Raman profiles. The absorption spectrum and the Raman, hyper-Rayleigh, and hyper-Raman excitation profiles are quantitatively simulated under the assumption that three excited electronic states contribute to the one- and two-photon absorption in this region. The transition centered near 400 nm is largely localized on the nitrophenyl group, while the transitions near 475 and 355 nm are more delocalized.     

Microsecond melting of a folding intermediate in a coiled-coil peptide, monitored by T-jump/UV Raman spectroscopy

A truncated version of the GCN4 coiled-coil peptide has been studied by ultraviolet resonance Raman (UVRR) spectroscopy with 197 nm excitation, where amide modes are optimally enhanced. Although the CD melting curve could be satisfactorily described with a two-state transition having Tm = 30 degrees C, singular value decomposition of the UVRR data yielded three principal components, whose temperature dependence implicates an intermediate form between the folded and unfolded forms, with formation and melting temperatures of 10 and 40 degrees C. Two alpha-helical amide III bands, at 1340 and 1300 cm(-1), melted out selectively at 10 and 40 degrees C, respectively, and are assigned to hydrated and unhydrated helical regions. The hydrated regions are proposed to be melted in the intermediate form, while the unhydrated regions are intact. Time-resolved UVRR spectra following laser-induced temperature jumps revealed two relaxations, with time constants of 0.2 and 15 mus. These are suggested to reflect the melting times of hydrated and unhydrated helices. The unhydrated helical region may be associated with a 14-residue "trigger" sequence that has been identified in the C-terminal half of GCN4. Dehydration of helices may be a key step in the folding of coiled-coils.     

Structural and vibrational characterization of the mono and dilithiated species derived from phenylacetonitrile in THF by infrared and Raman spectroscopy and density functional theory calculations

The structures and spectra of mono and dianionic species derived from PhCH2CN under the action of an organic base LHMDS or n-BuLi in THF solution have been investigated by vibrational spectroscopy and DFT calculations. The assignments previously proposed for the monoanion, the bridged, linear and dimeric monoanionic ion pairs compare well with the calculated ones. The addition of HMPA to a THF solution of PhCHCNLi leads to the formation of an HMPA solvated linear ion pair, distinguishable from the bridged ion pair by the wave number of the 8a v(CC) phenyl ring mode. The calculated structure of the phenylacetonitrile anion in the (PhCHCNLi, CH3Li) mixed dimer or 'Quadac' is very similar to that in the linear ion pair or in the dimer and to that observed by X-ray in (PhCHCNLi, [CH(CH9)2]2NLi) 'Quadac'. The structure of the anion is planar, indicating a charge delocalization from the benzene ring to the -CHCN group. The calculated spectra of the free, mono and dilithiated dianionic species compare well with the experimental ones of the species formed under addition of more than one equivalent of n-BuLi. The structure of the >C-C-CN2- group is very close to that of an imine. The v(CN) bands of the free, mono and dilithiated dianionic species are located at 1912, 1930 and 1890 cm(-1), respectively. The large wave number shift observed between the free monoanion and dianion (approximately 176 cm(-1)), in good accordance with the calculated one (170 cm(-1)) allows differentiating mono and dianionic species. The shifts observed for the 8a and 19a v(CC) phenyl ring modes, although much smaller, also allows discriminating the different species. These small shifts indicate a small variation of the electronic delocalization in the benzene ring in agreement with the calculations.     

Conversion of extradiol aromatic ring-cleaving homoprotocatechuate 2,3-dioxygenase into an intradiol cleaving enzyme

The intra- and extradiol subfamilies of catechol-adduct ring-cleaving dioxygenases each exhibit nearly absolute fidelity for the ring cleavage position. This is often attributed to the fact that the oxygen activation mechanism of intradiol dioxygenases utilizes Fe3+ while that of the extradiol enzymes employs Fe2+, but the subfamilies also differ in primary sequence, structural fold, iron ligands, and second sphere active site amino acid residues. Here, we examine the effects of the second sphere residue H200 in the active site of homoprotocatechuate 2,3-dioxygenase (2,3-HPCD), an extradiol-cleaving enzyme. It is shown that the H200F mutant enzyme catalyzes extradiol cleavage of the normal substrate, homoprotocatechuate (HPCA), but intradiol cleavage of the alternative substrate 2,3-dihydroxybenzoate (2,3-DHB) while in the Fe2+ oxidation state. Wild-type 2,3-HPCD catalyzes extradiol cleavage of both substrates. This is the first report of intradiol cleavage by an extradiol dioxygenase. It suggests that intradiol cleavage can occur with the iron in the Fe2+ state, with the iron ligand set characteristic of extradiol dioxygenases, and through a mechanism in which oxygen is activated by binding to the iron rather than directly attacking the substrate as in true intradiol dioxygenases. This indicates that substrate binding geometry and acid/base chemistry of second sphere residues play important roles in determining the course of the dioxygenase reaction.     

Enthalpic and entropic stages in alpha-helical peptide unfolding, from laser T-jump/UV Raman spectroscopy

The alpha-helix is a ubiquitous structural element in proteins, and a number of studies have addressed the mechanism of helix formation and melting in simple peptides. However, fundamental issues remain to be resolved, particularly the temperature (T) dependence of the rate. In this work, we report application of a novel kHz repetition rate solid-state tunable NIR (pump) and deep UV Raman (probe) laser system to study the dynamics of helix unfolding in Ac-GSPEA3KA4KA4-CO-D-Arg-CONH2, a peptide designed for helix stabilization in aqueous solution. Its T-dependent UV resonance Raman (UVRR) spectra, excited at 197 nm for optimal enhancement of amide vibrations, were decomposed into variable contributions from helix and coil spectra. The helix fractions derived from the UVRR spectra and from far UV CD spectra were coincident at low T but deviated increasingly at high T, the UVRR curve giving higher helix content. This difference is consistent with the greater sensitivity of UVRR spectra to local conformation than CD. After a laser-induced T-jump, the UVRR-determined helix fractions defined monoexponential decays, with time-constants of approximately 120 ns, independent of the final T (Tf = 18-61 degrees C), provided the initial T (Ti) was held constant (6 degrees C). However, there was also a prompt loss of helicity, whose amplitude increased with increasing Tf, thereby defining an initial enthalpic phase, distinct from the subsequent entropic phase. These phases are attributed to disruption of H-bonds followed by reorientation of peptide links, as the chain is extended. When Ti was raised in parallel with Tf (10 degrees C T-jumps), the prompt phase merged into an accelerating slow phase, an effect attributable to the shifting distribution of initial helix lengths. Even greater acceleration with rising Ti has been reported in T-jump experiments monitored by IR and fluorescence spectroscopies. This difference is attributable to the longer range character of these probes, whose responses are therefore more strongly weighted toward the H-bond-breaking enthalpic process.     

Experimental and Density Functional Theory Calculation Studies on Raman and Infrared Spectra of 1,1'-Binaphthyl-2,2'-diamine

The IR absorption, visible excited normal Raman, and UV-excited near-resonant Raman (UVRR) spectra of 1,1'-binaphthyl-2,2'-diamine (BINAM) were measured and analyzed. Density functional theory calculations were carried out to investigate its vibrational frequencies, infrared absorption, normal Raman, and near-resonance Raman intensities. The observed Raman and IR bands of BINAM were assigned with respect to the local vibrations of substituted 2-naphthylamine. Several Raman bands of BINAM were found selectively enhanced in the UVRR in comparison with the normal Raman spectrum. Possible excited state geometry distortion was discussed based on the resonance Raman intensity analysis.     

Characterization of the phenoxyl radical in model complexes for the Cu(B) site of cytochrome c oxidase: steady-state and transient absorption measurements, UV resonance raman spectroscopy, EPR spectroscopy, and DFT calculations for M-BIAIP

Physicochemical properties of the covalently cross-linked tyrosine-histidine-Cu(B) (Tyr-His-Cu(B)) unit, which is a minimal model complex [M(II)-BIAIPBr]Br (M = Cu(II), Zn(II)) for the Cu(B) site of cytochrome c oxidase, were investigated with steady-state and transient absorption measurements, UV resonance Raman (UVRR) spectroscopy, X-band continuous-wave electron paramagnetic resonance (EPR) spectroscopy, and DFT calculations. The pH dependency of the absorption spectra reveals that the pK(a) of the phenolic hydroxyl is ca. 10 for the Cu(II) model complex (Cu(II)-BIAIP) in the ground state, which is similar to that of p-cresol (tyrosine), contrary to expectations. The bond between Cu(II) and nitrogen of cross-linked imidazole cleaves at pH 4.9. We have successfully obtained UVRR spectra of the phenoxyl radical form of BIAIPs and have assigned bands based on the previously reported isotope shifts of Im-Ph (2-(1-imidazoyl)-4-methylphenol) (Aki, M.; Ogura, T.; Naruta, Y.; Le, T. H.; Sato, T.; Kitagawa, T. J. Phys. Chem. A 2002, 106, 3436-3444) in combination with DFT calculations. The upshifts of the phenoxyl vibrational frequencies for 8a (C-C stretching), 7a' (C-O stretching), and 19a, and the Raman-intensity enhancements of 19b, 8b, and 14 modes indicate that UVRR spectra are highly sensitive to imidazole-phenol covalent linkage. Both transient absorption measurements and EPR spectra suggest that the Tyr-His-Cu(B) unit has only a minor effect on the electronic structure of the phenoxyl radical form, although our experimental results appear to indicate that the cross-linked Tyr radical exhibits no EPR. The role of the Tyr-His-Cu(B) unit in the enzyme is discussed in terms of the obtained spectroscopic parameters of the model complex.     

Resonance Raman and semiempirical electronic structure studies of an odd-electron dinickel tetraiminoethylenedimacrocycle complex

Resonance Raman studies of Ni2TIED3+ (TIED = tetraiminoethylenedimacrocycle) reveal that many modes couple to the intense electronic transition centered at 725 nm, a feature that is nominally similar to the intense delocalized intervalence absorption bands observed in the same region for Fe2(TIED)L4(5+) and Ru2(TIED)L4(5+) (L is any of several axial ligands). Time-dependent spectral modeling of the Raman and absorption spectra for the nickel compound was undertaken to understand the electronic transition. We were unable to model the Raman and absorption spectra successfully with a single electronic transition, suggesting that the absorption band is made up of two overlapping transitions. Semiempirical electronic structure calculations corroborate the suggestion. Additionally, these calculations indicate that the transitions are in fact ligand-localized transitions, with little metal involvement and no charge-transfer character. Furthermore, the ground-state electronic structure is best described as an identical pair of NiII centers bridged by a radical anion rather than a three-site mixed-valence assembly. Previous EPR studies (McAuley and Xu, Inorg. Chem. 1992, 31, 5549) had indicated primarily ligand character for the radical. The assignments are consistent with the resonance Raman results where the dominant modes coupled to the transitions are assigned as totally symmetric bridge vibrations.     

IR,Raman, and UV/Vis Spectra of Corannulene for Use in Possible Interstellar Identification

The spectroscopic characterization of corannulene (C₂₀H₁₀) is carried out by several techniques. The high purity of the material synthesized for this study was confirmed by gas chromatography‐mass spectrometry (GC‐MS). During a high‐performance liquid chromatography (HPLC) process, the absorption spectrum of corannulene in the ultraviolet (UV) and visible (vis) ranges is obtained. The infrared (IR) absorption spectrum is measured in CsI pellets, and the Raman scattering spectrum is recorded for pure crystal grains. In addition to room temperature measurements, absorption spectroscopy in an argon matrix at 12 K is also performed in the IR and UV/Vis ranges. The experimental spectra are compared with theoretical Raman and IR spectra and with calculated electronic transitions. All calculations are based on the density functional theory (DFT), either normal or time‐dependent (TDDFT). Our results are discussed in view of their possible application in the search for corannulene in the interstellar medium.     

Resonance hyper-Raman excitation profiles of a donor-acceptor substituted distyrylbenzene: one-photon and two-photon states

Resonance Raman and resonance hyper-Raman spectra of the "push-pull" conjugated molecule 1-(4'-dihexylaminostyryl)-4-(4"-nitrostyryl)benzene in acetone have been measured at excitation wavelengths from 485 to 356 nm (two-photon wavelengths for the nonlinear spectra), resonant with the first two bands in the linear absorption spectrum. The theory of resonance hyper-Raman scattering intensities is developed and simplified using assumptions appropriate for intramolecular charge-transfer transitions of large molecules in solution. The absorption spectrum and the Raman, hyper-Rayleigh, and hyper-Raman excitation profiles, all in absolute intensity units, are quantitatively simulated to probe the structures and the one- and two-photon transition strengths of the two lowest-energy allowed electronic transitions. All four spectroscopic observables are reasonably well reproduced with a single set of excited-state parameters. The two lowest-energy, one-photon allowed electronic transitions have fairly comparable one-photon and two-photon transition strengths, but the higher-energy transition is largely localized on the nitrophenyl group while the lower-energy transition is more delocalized.