Raman Marker Research Articles

Influence of Meso Substituents on Electronic States of (Oxoferryl)porphyrin pi-Cation Radicals.

A series of (oxoferryl)porphyrin pi-cation radicals generated from porphyrins substituted at the meso positions with highly electron-withdrawing aryl groups has been characterized: tetrakis-5,10,15,20-(2,6-dichlorophenyl)-, 5-(2-chloro-6-nitrophenyl)-10,15,20-tris(2,6-dichlorophenyl)-, and 5-(2,6-dinitrophenyl)-10,15,20-tris(2,6-dichlorophenyl)porphyrins (porphyrins 1-3, respectively). The physical-chemical properties of the oxidized complexes of 1-3 are compared to those of two (oxoferryl)porphyrin pi-cation radical complexes substituted with electron-releasing aryl groups: tetramesitylporphyrin (TMP) and 2-iodotetramesitylporphyrin (2-iodoTMP). While all of the complexes examined show close correspondance in a number of spectroscopic parameters, some significant differences were observed. In contrast to observations for the oxidized complexes of TMP and 2-iodoTMP, the resonance Raman marker bands nu(2) and nu(11), which are indicators of symmetry state of porphyrin pi-cation radicals of 1-3, do not show the expected downfrequency shifts for oxidation to compound I analogs in a(2u) symmetry states. The upfield hyperfine NMR shifts of the pyrrole beta-proton signals of the compound I analogs of 1-3 are much larger than those for TMP and 2-iodoTMP. These data may be explained by admixture of some a(1u) character into the ground state of radical cations of 1-3, consistent with the hypothesis that electron-withdrawing meso substituents lower the energy of the a(2u) molecular orbital, favoring an a(1u) admixture.

Structure and dynamics of interstrand guanine association in quadruplex telomeric DNA.

Exchanges of the amino and imino protons in guanine quartets of telomeric DNA have been time-resolved by laser Raman spectroscopy. The Raman dynamic probe has been applied to parallel and antiparallel quadruplexes formed by the telomeric repeat of Oxytricha nova, d(T4G4)4, and to the highly thermostable parallel quadruplex formed by d(G12). Time-dependent Raman spectra of the d(G12) quadruplex reveal two characteristic exchange reactions for guanine N2 amino groups. At 10 degrees C, the pseudo-first-order exchange rates are kN2H(10 degrees C) = 5.7 x 10(-3) and k'N2H'(10 degrees C) = 1.2 x 10(-2) min-1, assignable to Hoogsteen-hydrogen-bonded and non-hydrogen-bonded N2 protons, respectively. These measurements provide the first quantitative determination of two kinetically distinct N2 amino proton exchange reactions in the guanine quartet and demonstrate that amino group rotation about the C2-N2 bond is highly restricted in the quadruplex. No exchange of guanine N1 imino sites occurs in d(G12) at 10 degrees C, and N1 exchange remains slow even at 95 degrees C [kN1H(95 degrees C) = 2.7 x 10(-2) min-1], indicating severe suppression of imino exchange in guanine quartets. For both parallel and antiparallel quadruplexes of d(T4G4)4, proton exchange rates decrease in the order thymine N3 imino > guanine N2 amino > guanine N1 imino. The rapid exchange of thymine N3 imino sites indicates that thymine quartets are not stabilized in Oxytricha quadruplexes. The protium-->deuterium exchange experiments also establish new guanine Raman band assignments. Importantly, the 1603 cm-1 band is due to in-plane bending of N1-H, while the 1644 cm-1 band involves scissoring of the N2 amino group. Accordingly, the 1603 and 1644 cm-1 bands are potentially valuable markers of hydrogen-bonding interactions specific to guanine imino and amino sites, respectively. The present findings also show that guanine hydrogen bonding and exchange dynamics are not interrelated in a simple manner. Despite extraordinary retardation of N1 imino proton exchange, Raman markers suggest that Hoogsteen-type guanine-guanine hydrogen bonding (N1-H...O=C6) is comparable in strength to hydrogen bonding of N1-H with water and, surprisingly, much weaker than hydrogen bonding between N1-H and the cytosine N3 acceptor of Watson-Crick B DNA.

Fingerprinting petroporphyrin structures with vibrational spectroscopy: II. Resonance Raman marker bands for the exocyclic rings of nickel tetrahydrobenzoporphyrins and nickel cycloalkanoporphyrins

The Q-band excited (568.2 nm) resonance Raman (RR) spectra are reported for a series of geologically significant nickel(II) complexes of etio-, cycloalkano-, and tetrahydro-benzoporphyrins in the frequency range from 600 to 1200 cm −1. Several patterns in the RR spectra of these complexes can be utilized as signatures for each petroporphyrin family. Useful marker bands for these classes of petroporphyrins are identified and distinguishing features of the spectra are given which can differentiate closely related isomers within a given class.

Polarized Raman spectra of oriented fibers of A DNA and B DNA: anisotropic and isotropic local Raman tensors of base and backbone vibrations

Polarized Raman spectra of oriented fibers of calf thymus DNA in the A and B conformations have been obtained by use of a Raman microscope operating in the 180 degrees back-scattering geometry. The following polarized Raman intensities in the spectral interval 200-1800 cm-1 were measured with both 514.5 and 488.0 nm laser excitations: (1) Icc, in which the incident and scattered light are polarized parallel to the DNA helical axis (c axis); (2) Ibb, in which the incident and scattered light are polarized perpendicular to c; and (3) Ibc and Icb, in which the incident and scattered light are polarized in mutually perpendicular directions. High degrees of structural homogeneity and unidirectional orientation were confirmed for both the A and B form fibers, as judged by comparison of the observed Raman markers and intensity anisotropies with measurements reported previously for oligonucleotide single crystals of known three-dimensional structures. The fiber Raman anisotropies have been combined with solution Raman depolarization ratios to evaluate the local tensors corresponding to key conformation-sensitive Raman bands of the DNA bases and sugar-phosphate backbone. The present study yields novel vibrational assignments for both A DNA and BDNA conformers and also confirms many previously proposed Raman vibrational assignments. Among the significant new findings are the demonstration of complex patterns of A form and B form indicator bands in the spectral intervals 750-900 and 1050-1100 cm-1, the identification of highly anisotropic tensors corresponding to vibrations of base, deoxyribose, and phosphate moieties, and the determination of relatively isotropic Raman tensors for the symmetrical stretching mode of phosphodioxy groups in A and B DNA. The present fiber results provide a basis for exploitation of polarized Raman spectroscopy to determine DNA helix orientation as well as to probe specific nucleotide residue orientations in nucleoproteins, viruses, and other complex biological assemblies.

A and Z canonical conformations in d(CnGCGn) crystals characterized by microFTIR and microRaman spectroscopies.

Two crystals d(C2GCG2) and d(C5GCG5) have been studied under microscope by Fourier transform ir spectroscopy and Raman spectroscopy. The x-ray diffraction study of the latter crystal had shown that the d(C5GCG5) sequence is the first DNA dodecamer known to adopt a canonical A conformation [N. Verdaguer, J. Aymami, D. Fernandez-Forner, I. Fita, M. Coll, T. Huynh-Dinh, J. Igolen, and J. A. Subirana (1991) Journal of Molecular Biology, Vol. 221, pp. 623-635]. Characteristic ir marker bands and Raman marker peaks of the A conformation have thus been obtained and are compared with previously proposed assignments correlated to fiber diffraction x-ray results obtained on polymers. The d(C2GCG2) sequence crystal had previously been studied in an intermediate form between B and Z [L. Urpi, J. P. Ridoux, J. Liquier, N. Verdagner, I. Fita, J. A. Subirana, F. Iglesias, T. Huynh-Dinh, J. Igolen, and E. Taillandier (1989) Nucleic Acids Research, Vol. 17, pp. 6669-6679]. In this paper we present results obtained from a crystal with this oligonucleotide in Z conformation. The effect of the crystallization conditions on the geometry of the obtained oligomer helix is discussed. The influence of the addition, to the central tetramer CGCG, of dCn stretches (at the 5' end) and dGn stretches (at the 3' end) of different lengths, on the conformational flexibility of the nucleic acid, is considered.

Structural Studies of the Enveloped dsRNA Bacteriophage θ6 of Pseudomonas syringae by Raman Spectroscopy: II. Nucleocapsid Structure and Thermostability of the Virion, Nucleocapsid and Polymerase Complex

Structures and thermostabilities of the double-stranded (ds) RNA bacteriophage θ6 and of its isolated nucleocapsid-polymerase complex (nucleocapsid core) and dsRNA components have been investigated by Raman spectroscopy. The spectra show that proteins of the θ6 virion are collectively deficient in β-sheet secondary structure. In particular, the major protein (P8) of the outer spherical shell of the θ6 nucleocapsid exhibits a secondary structure dominated largely by α-helix and irregular conformations. The absence of appreciable β-structure in the P8 subunit suggests a tertiary conformation lacking the β-barrel motif common to subunits of most other spherical viral capsids. In addition, the Raman spectra show that subunits of the dodecahedral nucleocapsid core are also predominantly α-helical. The results thus indicate a largely α-helical secondary structure for the major subunit (P1) of the θ6 nucleocapsid core, as well as for the P8 subunit of the outer spherical shell.Using Raman difference spectroscopy, we demonstrate that proteins of the nucleocapsid core (P1, P2, P4 and P7) interact extensively with the packaged θ6 RNA genome, and further, that conformational stability of the packaged RNA is reduced upon removal from the core. Also, we find that proteins of the θ6 nucleocapsid are significantly more thermostable than proteins of the viral membrane envelope, which are reported in the accompanying paper (Li et al., 1993). The present results suggest that both the architectural principles and modes of protein-RNA interaction in the θ6 virion differ fundamentally from those of icosahedral single-stranded RNA viruses.Both Raman and circular dichroism spectra indicate that the dsRNA genome of θ6 is an A-form structure. The Raman marker bands signify the presence only of C3′-endo/anti nucleoside conformers. The Raman signature of dsRNA, revealed in the spectrum of the θ6 genome, is discussed here as a model for assessing base-pairing and base-stacking interactions in other ribonucleoprotein assemblies.

Demonstration of Z-d(5BrCGAT5BrCG) and B-d(CGCGATCGCG) form crystal structures in DNA-cobalt hexammine complexes by Kr 647.1 nm excitation of Raman spectra.

Cobalt hexammine [Co(NH3)6(3+)] is an efficient DNA complexing agent which significantly perturbs nucleic acid secondary structure. We have employed red excitation (647.1 nm) from a krypton laser to obtain Raman spectra of the highly colored complexes formed between cobalt hexammine and crystals of the DNA oligomers, d(5BrCGAT5BrCG) and d(CGCGATCGCG), both of which incorporate out-of-alternation pyrimidine/purine sequences. The Co(NH3)6(3+) complex of d(5BrCGAT5BrCG) exhibits a typical Z-form Raman signature, similar to that reported previously for the alternating d(CGCGCG) sequence. Comparison of the Raman bands of d(5BrCGAT5BrCG) with those of other oligonucleotide and polynucleotide structures suggests that C3'-endo/syn and C3'-endo/anti thymidines may exhibit distinctive nucleoside conformation markers, and tentative assignments are proposed. The Raman markers for C2'-endo/anti adenosine in this Z-DNA are consistent with those reported previously for B-DNA crystals containing C2'-endo/anti dA. Raman bands of the cobalt hexammine complex of d(CGCGATCGCG) are those of B-DNA, but with significant differences from the previously characterized B-DNA dodecamer, d(CGCAAATTTGCG). The observed differences suggest an unusual deoxyguanosine conformer, possibly related to a previously characterized structural intermediate in the B-->Z transition. The present results show that crystallization of d(CGCGATCGCG) in the presence of cobalt hexammine is not alone sufficient to induce the left-handed Z-DNA conformation. This investigation represents the first application of off-resonance Raman spectroscopy for characterization of highly chromophoric DNA and illustrates the feasibility of the Raman method for investigating other structurally perturbed states of DNA-cobalt hexammine complexes.

Conformation of d(GGGATCCC)2 in crystals and in solution studied by X-ray diffraction, Raman spectroscopy and molecular modelling.

In the crystal, d(GGGATCCC)2 forms an A-DNA double helix as known from a single crystal X-ray diffraction study. Accordingly, in the Raman spectra of crystals the A-family marker bands at 664, 705, 807 and 1101 cm-1 and the spectral characteristics in the region 1200 to 1500 cm-1 clearly demonstrate the A-form as the dominant conformation. Bands at 691, 850, and 1080 cm-1, however, indicate that a minor fraction of the octamer molecules in the crystal is in an unusual, still not unequivocally identified conformation possibly belonging to the B-family. In solution, the octamer is in B-like conformation as shown by the presence of B-DNA Raman marker bands at 685, 837, 1094 and 1421 cm-1. Molecular modelling techniques lead to three structures with slightly different B-form geometries as the lowest energies models when a sigmoidal dielectric function with the bulk dielectric constant epsilon = 78 and the value q = -0.5e for the effective phosphate charges was used in the calculations. An A-form structure bearing a strong resemblance to the experimentally determined crystal structure becomes the lowest energy model structure when the electrostatic parameters are changed to epsilon = 30 and q = -0.25e, respectively.

Ultraviolet resonance Raman marker bands of the right to left helix structure transitions in DNA and polynucleotide model compounds.

The right to left helix structural transition in purine-pyrimidine alternating copolymers has been extensively studied by vibrational spectroscopies, amongst many other experimental approaches. Here, the use of resonance Raman spectroscopy in the ultraviolet region (223-, 257- and 281 nm excitation wavelengths) to monitor such structural changes is reviewed in the light of new results obtained on poly(dA-dC).poly(dG-dT) on one hand, and the previous results obtained on poly(dG-dC)2, poly(dA-dT)2 and natural DNA (Chicken erythrocytes) on the other. It is now possible to define B----Z transition marker bands involving the proper bases, which show a similar behaviour on structural transition whatever the composition of alternating purine-pyrimidine sequences: the 1580- and 1487 cm-1 lines of the purines, the 1486- and 1294 cm-1 lines of the pyrimidines are good markers in the vibrational spectra recorded at various UV excitation wavelengths.

Conformations, interactions, and thermostabilities of RNA and proteins in bean pod mottle virus: investigation of solution and crystal structures by laser Raman spectroscopy.

We report and interpret laser Raman spectra of the three virion components of bean pod mottle virus (BPMV). The top component of BPMV is an empty capsid; middle and bottom components package the RNA2 and RNA1 genome segments, respectively. All components were investigated as both single crystals and aqueous solutions, the latter over wide ranges of temperature and ionic strength. The isolated RNA2 molecule of BPMV middle component was also investigated in both H2O and D2O solutions. The results permit assessment of RNA and protein structures, their mutual interactions in the virions, and their conformational thermostabilities and comparison of these structural characteristics for solution and crystal states of the particles. The principal findings of this study are (i) The extent of ordered A-form backbone (74%) and of base pairing (38% AU + 22% GC) in unpackaged (aqueous) RNA2 are significantly altered by packaging. The A-form secondary structure of RNA2 is increased by 12 +/- 4%, and guanine base interactions are also substantially increased with packaging. (ii) The thermostability of Raman-monitored secondary structure of unpackaged RNA2 (Tm approximately 43 degrees C) is greatly increased in the packaged state (Tm approximately 53 degrees C). This increase corresponds to a stabilization of the A-form backbone geometry in 15 +/- 5% of genome nucleotides. (iii) Packaging of RNA2 in the middle component stabilizes subunit-subunit interactions of the capsid, as evidenced by a thermal denaturation temperature Td approximately 65 degrees C for the virion, compared with Td approximately 55 degrees C for the empty capsid. (iv) Raman marker-band shifts implicate the purine 7N sites of RNA2 and aromatic side chains of subunits as the principal targets for RNA-subunit interaction. (v) At the conditions of the present experiments (8 degrees C, pH approximately 7, moderate ionic strength), the subunit secondary structures observed for solutions of the top, middle, and bottom components are indistinguishable by Raman spectroscopy from secondary structures observed for corresponding crystalline samples. (vi) On the other hand, side chains of subunits in the top component (empty capsid) yield significantly different Raman intensities in crystalline and solution states. These differences are interpreted as the result of changes in a small number of side-chain environments between crystal and solution. (vii) Similarly, small differences exist between RNA Raman markers of crystalline and aqueous virions, which are attributed to altered environments of nucleotide residues and to a small increase in the amount of A-form backbone geometry upon going from the crystal to the solution.(ABSTRACT TRUNCATED AT 400 WORDS)

Raman spectroscopy of filamentous bacteriophage Ff (fd, M13, f1) incorporating specifically-deuterated alanine and tryptophan side chains. Assignments and structural interpretation

Structural interpretation of the Raman spectra of filamentous bacteriophages is dependent upon reliable assignments for the numerous Raman vibrational bands contributed from coat protein and packaged DNA of the virion. To establish unambiguous assignments and facilitate structural conclusions derived from them, we have initiated a systematic study of filamentous bacteriophage Ff (fd, f1, M13) incorporating protein subunits with specifically deuterated amino-acid side chains. Here, we report and interpret the Raman spectra of fd virions which incorporate: (a) a single deuterio-tryptophan residue per coat protomer [fd(Wd5)], (b) ten deuterio-alanines per protomer [fd(10Ad3)], and (c) both deuterio-tryptophan and deuterio-alanine [fd(Wd5 + 10Ad3)]. The unambiguous assignment of coat protein Raman bands in normal and deuterated isotopomers of fd establishes the validity of earlier empirical assignments of many key Raman markers, including those of packaged ssDNA (Thomas et al., 1988). Present results confirm that deoxyguanosine residues of the packaged ssDNA molecule depart from the usual C2'-endo/anti conformation characteristic of protein-free DNA in aqueous solution, although C2'-endo/anti conformers of thymidine are not excluded by the data. The combined results obtained here on normal fd, and on fd incorporating deuterio-tryptophan [fd(Wd5) and fd(Wd5 + 10Ad3)], show also that the microenvironment of the single tryptophan residue per coat protomer (W26) can be clearly deduced as follows: (a) The indole 1-NH donor group of each protomer in fd forms a moderately strong hydrogen bond, most likely to a hydroxyl oxygen acceptor. (b) The planar indole ring exists in a hydrophilic environment. (c) The torsion angle describing the orientation of the indole ring (C3-C2 linkage) with respect to the side-chain (C alpha-C beta bond) is unusually large, i.e., magnitude of X2,1 approximately 120 degrees. With respect to alanine isotopomers, the present results show that alanine residues, and possibly other methyl-containing side chains, are significant contributors to the fd Raman spectrum. The present study provides new information on protomer side chains of fd and demonstrates a Raman methodology which should be generally useful for investigating single-site interactions and macromolecular conformations in other nucleoprotein assemblies.

DNA recognition by the helix-turn-helix motif: investigation by laser Raman spectroscopy of the phage .lambda. repressor and its interaction with operator sites OL1 and OR3

The lambda repressor provides a model system for biophysical studies of DNA recognition by the helix-turn-helix motif. We describe laser Raman studies of the lambda operator sites OL1 and OR3 and their interaction with the DNA-binding domain of lambda repressor (residues 1-102). Raman spectra of the two DNA sites exhibit significant differences attributable to interstrand purine-purine steps that differ in the two oligonucleotides. Remarkably, the conformation of each operator is significantly and specifically altered by repressor binding. Protein recognition, which involves hydrogen-bond formation and hydrophobic contacts in the major groove, induces subtle changes in DNA Raman bands of interacting groups. These include (i) site-specific perturbations to backbone phosphodiester geometry at AT-rich domains, (ii) hydrophobic interaction at thymine 5CH3 groups, (iii) hydrogen bonding to guanine 7N and 6C = O acceptors, and (iv) alterations in sugar pucker within the C2'-endo (B-DNA) family. These perturbations differ between aqueous OL1 and OR3 complexes of repressor, indicating that protein binding in solution determines the precise DNA conformation. The overall structure of the lambda domain is not greatly perturbed by binding to either OL1 or OR3, in accord with X-ray studies of other complexes. However, Raman markers indicate a change in hydrogen bonding of the OH group of tyrosine-22, which is a hydrogen-bond acceptor in the absence of DNA but a combined donor and acceptor in the OL1 complex; yet, Y22 hydrogen bonding is not altered in forming the OR3 complex. The present results demonstrate qualitatively different and distinguishable modes of interaction of the lambda repressor DNA-binding domain with operators OL1 and OR3 in solution. This application of laser Raman spectroscopy to a well-characterized system provides a prototype for future Raman studies of other DNA-binding motifs under physiological conditions.

Secondary structure of the double‐stranded DNA genome of bacteriophage T7 in packaged, underpackaged and unpackaged states

AbstractDouble‐stranded DNA exhibits prominent Raman bands in the spectral region 600–900 cm−1, authenticated by x‐ray crystallography as diagnostic of the macromolecular secondary structure. Since the DNA Raman fingerprint can be measured in the presence of protein, Raman spectroscopy was employed to investigate the secondary structure of dsDNA packaged within the protein coat of bacteriophage T7. Comparison of the Raman spectrum of deproteinized DNA with that of wild‐type T7 phage (52% DNA, 48% protein) reveals a packaging‐associated shift of the principal BDNA Raman marker (the band of the 825–840 cm−1 interval which is a sensitive indicator of phosphodiester geometry), without shifts of other conformation sensitive Raman markers (the bands of the 600–780 cm−1 interval which indicate sugar puckers and glycosyl torsions). The Raman marker for the protein‐free state occurs at 836 cm−1, typical of B‐form DNA of heterogeneous sequence; the marker for the packaged state occurs at 825 cm−1, characteristic of a modified B‐form DNA observed previously for specific sequences. These findings indicate different B‐form variants for dsDNA in packaged and protein‐free states. The modified B‐form secondary structure of packaged DNA is maintained when the virion is thermally burst to release the genome without removal of proteins.Comparison of the Raman spectrum of the wild‐type T7 virion with that of a deletion mutant packaging 8.4% less DNA than wild‐type (underpackaged T7), reveals the same packaging‐associated shift of the principal B‐form marker (825 cm−1) vis‐à‐vis protein‐free DNA (836 cm−1), and additional evidence of small but significant differences between phosphodiester geometries in packaged and underpackaged states. These differences are explained by a marginally (<5%) greater proportion of non‐B backbone geometry in wild‐type T7. Both wild‐type and mutant T7 exhibit Raman markers of the C2′‐endo/anti nucleoside conformations exclusively. The results are interpreted in terms of the formation of increasing amounts of non B‐form phosphodiester geometry with increasing density of DNA packaging. The data are consistent with an excess of <5% A‐type phosphodiester geometry in packaged versus underpackaged states, and 10 ± 4% excess A‐type phosphodiester geometry in packaged versus protein‐free states. Based on Raman amide I and amide III markers, the protein structures and protein‐DNA interactions in wild‐type T7 are indistinguishable from those in the T7 deletion mutant.

Structural study of the lipid-containing bacteriophage PRD1 and its capsid and DNA components by laser Raman spectroscopy

The Raman spectrum of a virus contains the structural signature of each of its molecular components (Thomas, 1987). We report the first Raman spectrum obtained from an intact, lipid-containing virus--the icosahedral bacteriophage PRD1--and show that this spectrum contains characteristic structure markers for the major capsid protein, the packaged double-stranded DNA genome, and the viral membrane which resides between the capsid and DNA. We find that the packaged genome of PRD1 exhibits Raman markers typical of the B-DNA secondary structure. Comparison of the Raman spectrum of the packaged DNA with that of protein-free DNA extracted from the virion shows further that the B-form secondary structure is not significantly perturbed by packaging in the virion. The Raman signature of the PRD1 membrane, monitored within the virion at 4 degrees C, is that of a phospholipid liquid-crystalline phase. The PRD1 capsid, which comprises several hundred copies of the major coat protein P3 (product of viral gene III) and a few copies of minor proteins, incorporates P3 capsomers predominantly in the beta-sheet conformation. The beta-sheet structure of P3 is maintained in the fully assembled PRD1 virion, as well as in the empty capsid. The present results demonstrate the feasibility of obtaining structural information from the three different classes of biomolecules--nucleic acid, protein, and lipid--which constitute a membrane-lined virus particle. Our results also demonstrate that the coat protein and double-stranded DNA components of a lipid-containing bacteriophage share many structural features in common with bacteriophage lacking a lipid membrane.

Effect of the G.T mismatch on backbone and sugar conformations of Z-DNA and B-DNA: analysis by Raman spectroscopy of crystal and solution structures of d(CGCGTG) and d(CGCGCG).

The Z-DNA crystal structures of d(CGCGTG) and d(CGCGCG) are compared by laser Raman spectroscopy. Raman bands originating from vibrations of the phosphodiester groups and sensitive to the DNA backbone conformation are similar for the two structures, indicating no significant perturbation to the Z-DNA backbone as a result of the incorporation of G.T mismatches. Both Z structures also exhibit Raman markers at 625 and 670 cm-1, assigned respectively to C3'-endo/syn-dG (internal) and C2'-endo/syn-dG conformers (3' terminus). Additional Raman intensity near 620 and 670 cm-1 in the spectrum of the d(CGCGTG) crystal is assigned to C4'-exo/syn-dG conformers at the mismatch sites (penultimate from the 5' terminus). A Raman band at 1680 cm-1, detected only in the d(CGCGTG) crystal, is assigned to the hydrogen-bonded dT residues and is proposed as a definitive marker of the Z-DNA wobble G.T pair. For aqueous solutions, the Raman spectra of d(CGCGTG) and d(CGCGCG) are those of B-DNA, but with significant differences between them. For example, the usual B-form marker band at 832 cm-1 in the spectrum of d(CGCGTG) is about 40% less intense than the corresponding band in the spectrum of d(CGCGCG), and the former structure exhibits a companion band at 864 cm-1 not observed for d(CGCGCG). The simplest interpretation of these results is that the conventional B-form OPO geometry occurs for only 6 of the 10 OPO groups of d(CGCGTG). The remaining four OPO groups, believed to be those at or near the mismatch site, are in an "unusual B" conformation which generates the 864 cm-1 band.(ABSTRACT TRUNCATED AT 250 WORDS)

Structural Characterization of Cytochrome c Peroxidase by Resonance Raman Scattering

Resonance Raman scattering studies are reported on freshly prepared and aged ferric, ligand-free ferrous, and CO-bound ferrous cytochrome c peroxidase. The ferric form of the fresh enzyme has a heme which is penta-coordinate high spin, independent of buffer over the pH range 4.3-7, as determined by well established Raman marker lines. The aged enzyme displays a mixture of spin and coordination states, but it can be stabilized in the penta-coordinate high spin form in the presence of phosphate. These results can be accounted for by considering the size of the channel (6 A wide, 11 A long) between the distal side of the heme and the outer surface of the protein. A phosphate ion may be accommodated in this channel resulting in the stabilization of the distal heme pocket. The ferrous cytochrome c peroxidase in both the ligand-free and CO-bound states has an acidic and an alkaline form. The acidic form has the characteristic spectral features of peroxidases: a high frequency iron-histidine stretching mode (248 cm-1), a high frequency Fe-CO stretching mode (537 cm-1), and a low frequency C-O stretching mode (1922 cm-1). At alkaline pH these frequencies become similar to those of hemoglobin and myoglobin, with the corresponding modes located at 227, 510, and 1948 cm-1, respectively. We attribute the acid/alkaline transition in the ferrous forms of cytochrome c peroxidase to a rearrangement mainly of the proximal side of the heme, culminating in a change of steric interactions between the proximal histidine and the heme or of the hydrogen bonding network involving the proximal histidine. The new data presented here reconcile many inconsistencies reported in the past.

ChemInform Abstract: Axial Ligation‐Induced Structural Changes in Nickel Hydrocorphinoids Related to Coenzyme F430 Detected by Raman Difference Spectroscopy.

Raman difference spectroscopy provides a structural probe of the state of axial ligation of Ni(II) in nickel corphinoids of the type occurring in coenzyme F/sub 430/ of the methylreductase enzyme of methanogenic bacteria. The two model nickel corphinoids investigated, which have all of the structural features of the natural chromophore, were synthesized by A. Faessler, A. Pfaltz, and A. Eschenmoser. Raman lines analogues to the core-size marker lines of metalloporphyrins are identified for the Ni corphinoids, and, just as for the Ni porphyrins, the frequencies for the group of Raman marker lines characterize the 4-, 5-, and 6-coordinate complexes. Further, complexes with two strong nitrogenous bases (e.g., piperidine) can be distinguished from complexes with weak basic ligands (e.g., methanol) on the basis of the frequencies of these Raman lines. Finally, Raman spectra of polycrystalline samples of the Ni corphinoid and its monoisothiocyante complex show that the structures existing in solution differ markedly from the structures in the crystalline state. The Raman markers will be useful in elucidating the structure of coenzyme F/sub 430/ and its interaction with the nearby protein environment in component C of the methyl-S-coenzyme-M reductase system.

Axial ligation-induced structural changes in nickel hydrocorphinoids related to coenzyme F430 detected by Raman difference spectroscopy

Raman difference spectroscopy provides a structural probe of the state of axial ligation of Ni(II) in nickel corphinoids of the type occurring in coenzyme F/sub 430/ of the methylreductase enzyme of methanogenic bacteria. The two model nickel corphinoids investigated, which have all of the structural features of the natural chromophore, were synthesized by A. Faessler, A. Pfaltz, and A. Eschenmoser. Raman lines analogues to the core-size marker lines of metalloporphyrins are identified for the Ni corphinoids, and, just as for the Ni porphyrins, the frequencies for the group of Raman marker lines characterize the 4-, 5-, and 6-coordinate complexes. Further, complexes with two strong nitrogenous bases (e.g., piperidine) can be distinguished from complexes with weak basic ligands (e.g., methanol) on the basis of the frequencies of these Raman lines. Finally, Raman spectra of polycrystalline samples of the Ni corphinoid and its monoisothiocyante complex show that the structures existing in solution differ markedly from the structures in the crystalline state. The Raman markers will be useful in elucidating the structure of coenzyme F/sub 430/ and its interaction with the nearby protein environment in component C of the methyl-S-coenzyme-M reductase system.

Metalloporphyrin core size resonance Raman marker bands revisited: implications for the interpretation of hemoglobin photoproduct Raman frequencies

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMetalloporphyrin core size resonance Raman marker bands revisited: implications for the interpretation of hemoglobin photoproduct Raman frequenciesN. Parthasarathi, C. Hansen, S. Yamaguchi, and T. G. SpiroCite this: J. Am. Chem. Soc. 1987, 109, 13, 3865–3871Publication Date (Print):June 1, 1987Publication History Published online1 May 2002Published inissue 1 June 1987https://doi.org/10.1021/ja00247a009RIGHTS & PERMISSIONSArticle Views567Altmetric-Citations183LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (927 KB) Get e-Alerts Get e-Alerts

Heme-linked ionizations of myeloperoxidase detected by Raman difference spectroscopy. A comparison with plant and yeast peroxidases

The pH-dependence of the oxidation state marker line v4 of human leucocyte myeloperoxidase is determined in the absence of chloride using Raman difference spectroscopy (RDS). A transition in the frequency of v4 with pK of 4.2 +/- 0.3 is found. The pK compares favorably with that previously determined by spectrophotometric titration and kinetic studies. The shift in v4 across the transition is -1.3 cm-1. The shift in v4 and other Raman marker lines indicates enhanced pi charge in the chlorin ring below the transition. The low frequencies of the oxidation state marker lines indicate that a structural change occurs near the chromophore, which results in the formation of a more pi-charge donating protein environment for the chlorin ring at low pH. The Raman results are discussed in terms of a proposed catalytic control mechanism based on charge stabilization of the energy of ring charge-depleted ferryl intermediates of the reaction with peroxide. The myeloperoxidase findings are compared with similar RDS results for ferrous horseradish peroxidase and ferric cytochrome c peroxidase.