Conformational studies on the pyridoxal Schiff bases of polypeptides

The pyridoxal Schiff bases of the polypeptides poly(L -lysine), poly(L -ornithine), and poly(L -α,γ-diaminobutyric acid) were prepared and investigated in water/methanol by CD spectra and equilibrium dialysis experiments. Only the poly(L -α,γ-diaminobutyric acid) derivative is characterized by a relevant optical activity similar to that found in pyridoxal enzymes. The stereospecific interactions between the pyridoxylideneimine group and the polypeptide chain prevent the hydrolysis reaction of the aldimine bond.     

Resonance Raman spectroscopy

Resonance Raman spectroscopy may yield precise information on the conformation of, and on the interactions assumed by, the chromophores involved in the first steps of the photosynthetic process, whether isolated in solvents, embedded in soluble or membrane proteins, or, as shown recently, in vivo. By making use of this technique, it is possible, for instance, to relate the electronic properties of these molecules to their structure and/or the physical properties of their environment, or to determine subtle changes of their conformation associated with regulatory processes. After a short introduction to the physical principles that govern resonance Raman spectroscopy, the information content of resonance Raman spectra of chlorophyll and carotenoid molecules is described in this review, together with the experiments which helped in determining which structural parameter each Raman band is sensitive to. A selection of applications of this technique is then presented, in order to give a fair and precise idea of which type of information can be obtained from its use in the field of photosynthesis.     

Conformation of lanthanide complexes with Schiff bases of pyridoxal and dipeptides

In order to simulate the active centres of pyridoxal enzymes, lanthanide complexes of pyridoxal-dipeptide (gly-asn) azomethines were investigated by means of NMR 1H and optical spectroscopy. NMR spectra were shown to correspond to conformation averaged over two positions of the ion. The ion was bound to the oxygen atom of PL and nitrogen atom of azomethine. In addition, it coordinates the oxygen atom of the peptide bond or the asparagine carboxyl.     

Resonance Raman spectroscopy of polynucleotides

Resonance Raman Spectroscopy allows a selective study of the bases of DNA and therefore of the interactions of these bases with ligands. This technique is also sensitive to structural modifications. We show here that, first, the structures of native poly(dA-dT).poly(dA-dT) and poly(dA).poly(dT) are not the same and that, secondly, it is possible to characterize the B----Z transition of poly(dG-dC).poly(dG-dC). The study of the Raman hypochromism during the thermal denaturation of the polynucleotides reveals that the stacking of the adenines in poly(dA).poly(dT) is near that observed in poly(rA) but differs of this stacking in poly(dA-dT).poly(dA-dT). The enhancement of the intensity of the guanine line at 1193 cm-1 and of the cytosine lines at 780 cm-1, 1 242 cm-1 and 1268 cm-1 as well as the shift of the guanine line at low frequency should allow to characterize a small proportion of base pairs in Z form in any DNA.     

The oxidation of Schiff bases of pyridoxal and pyridoxal phosphate with amino acids by manganous ions and peroxidase

1. Oxygen was taken up rapidly when pyridoxal or pyridoxal phosphate was added to mixtures of pea-seedling extracts and Mn(2+) ions. 2. The increases in total oxygen uptake were proportional to the pyridoxal or pyridoxal phosphate added and were accompanied by the disappearance of these compounds. 3. In addition to Mn(2+) ions, the reactions depended on two factors in the extracts, a thermolabile one in the non-diffusible material and a thermostable one in the diffusate; these factors could be replaced in the reactions by horse-radish peroxidase (donor-hydrogen peroxide oxidoreductase, EC 1.11.1.7) and amino acids respectively. 4. When pyridoxal phosphate was added to mixtures of amino acids and Mn(2+) ions oxygen uptake was rapid after a lag period of 30-90min.; the lag period was shortened to a few minutes by peroxidase, particularly in the presence of traces of p-cresol, or by light. 5. When pyridoxal replaced pyridoxal phosphate relatively high concentrations were required and peroxidase had only a small activating effect. 6. Pyridoxal or pyridoxal phosphate disappeared during the reactions and carbon dioxide and ammonia were formed. 7. With phenylalanine as the amino acid present, benzaldehyde was identified as a reaction product. 8. It is suggested that the reactions are oxidations of the Schiff bases formed between pyridoxal or pyridoxal phosphate and amino acids, mediated by a manganese oxidation-reduction cycle, and resulting in oxidative decarboxylation and deamination of the amino acids.     

Resonance Raman spectroscopy in malaria research

In recent years, the field of Raman spectroscopy has witnessed a surge in technological development, with the incorporation of ultrasensitive, charge-coupled devices, improved laser sources and precision Rayleigh-filter systems. This has led to the development of sensitive confocal micro-Raman spectrometers and imaging spectrometers that are capable of obtaining high spatial-resolution spectra and images of subcellular components within single living cells. This review reports on the application of resonance micro-Raman spectroscopy to the study of malaria pigment (hemozoin), a by-product of hemoglobin catabolization by the malaria parasite, which is an important target site for antimalarial drugs. The review aims to briefly describe recent studies on the application of this technology, elucidate molecular and electronic properties of the malaria pigment and its synthetic analog β-hematin, provide insight into the mechanism of hemozoin formation within the food vacuole of the parasite, and comment on developing strategies for using this technology in drug-screening protocols.     

Resonance Raman spectroscopy in malaria research

In recent years, the field of Raman spectroscopy has witnessed a surge in technological development, with the incorporation of ultrasensitive, charge-coupled devices, improved laser sources and precision Rayleigh-filter systems. This has led to the development of sensitive confocal micro-Raman spectrometers and imaging spectrometers that are capable of obtaining high spatial-resolution spectra and images of subcellular components within single living cells. This review reports on the application of resonance micro-Raman spectroscopy to the study of malaria pigment (hemozoin), a by-product of hemoglobin catabolization by the malaria parasite, which is an important target site for antimalarial drugs. The review aims to briefly describe recent studies on the application of this technology, elucidate molecular and electronic properties of the malaria pigment and its synthetic analog beta-hematin, provide insight into the mechanism of hemozoin formation within the food vacuole of the parasite, and comment on developing strategies for using this technology in drug-screening protocols.     

Conformational studies on Cu(II) polycomplexes of pyridoxal Schiff bases of polypeptides

Structures of Cu(II) complexes of pyridoxal Schiff bases with poly(L -lysine), poly(L -ornithine), and poly(L -α,γ-diaminobutyric acid) were investigated by absorption spectra, CD, and conformational analysis. Although the polypeptides retain their typical right-handed α-helical conformation, opposite Cotton effects were found for the poly(L -lysine) and poly(L -ornithine) polycomplexes in the whole range of wavelengths from 600 to 250 nm. As in the analogous derivatives of salicyladehyde, this effect seems to be due to a stereospecific binding of the square planar Cu(II)-bis-pyridoxylideneimine group to the α-helical matrix. Circular dichroism spectrum of poly(L -α,γ-diaminobutyric acid) polycomplex is similar to that found for poly(L -lysine) derivative, but indicates large tetrahedral distortion of the square-planar coordination of copper ion.     

Resonance Raman spectroscopy of iron-oxygen vibrations in hemerythrin

Resonance Raman spectra are reported for oxyhemerythrin and 15 anionic adducts of methemerythrin. All methemerythrin derivatives except sulfidomethemerythrin contain a Raman band near 510 cm^?1 which is assigned to an iron-oxygen stretching vibration. The effect of H₂¹⁸O on the frequency of this vibration was studied extensively. On the basis of the exchange results, the vibration is assigned to OH^?, H₂O, or a μ-oxo bridge between the irons.     

Resonance Raman spectroscopy of carotenoids in Photosystem I particles

Low-temperature resonance Raman (RR) spectroscopy was used for the first time to study the spectral properties, binding sites and composition of major carotenoids in spinach Photosystem I (PSI) particles. Excitation was provided by an argon ion laser at 457.9, 476.5, 488, 496.5, 502 and 514.5 nm. Raman spectra contained the four known groups of bands characteristic for carotenoids (called from nu(1) to nu4). Upon 514.5, 496.5 and 476.5 nm excitations, the nu(1)-nu(3) frequencies coincided with those established for lutein. Spectrum upon 502-nm excitation could be assigned to originate from violaxanthin, at 488 nm to 9-cis neoxanthin, and at 457.9 nm to beta-carotene and 9-cis neoxanthin. The overall configuration and composition of these bound carotenoid molecules in Photosystem I particles were compared with the composition of pigment extracts from the same PSI particles dissolved in pyridine, as well as to configuration in the main chlorophyll a/b light-harvesting protein complex of photosystem II. The absorption transitions for lutein, violaxanthin and 9-cis neoxanthin in spinach photosystem I particles are characterized, and the binding sites of lutein and neoxanthin are discussed. Resonance Raman data suggest that beta-carotene molecules are also present in all-trans and, probably, in 9-cis configurations.     

Resonance Raman spectroscopy of octopus rhodopsin and its photoproducts

We report here the resonance Raman spectra of octopus rhodopsin and its photoproducts, bathorhodopsin and acid metarhodopsin. These studies were undertaken in order to make comparisons with the well-studied bovine pigments, so as to understand the similarities and the differences in pigment structure and photochemical processes between vertebrates and invertebrates. The flow method was used to obtain the Raman spectrum of rhodopsin at 13 degrees C. The bathorhodopsin spectrum was obtained by computer subtraction of the spectra containing different photostationary mixtures of rhodopsin, isorhodopsin, hypsorhodopsin, and bathorhodopsin, obtained at 12 K using the pump-probe technique and from measurements at 80 K. Like their bovine counterparts, the Schiff base vibrational mode appears at approximately 1660 cm-1 in octopus rhodopsin and the photoproducts, bathorhodopsin and acid metarhodopsin, suggesting a protonated Schiff base linkage between the chromophore and the protein. Differences between the Raman spectra of octopus rhodopsin and bathorhodopsin indicate that the formation of bathorhodopsin is associated with chromophore isomerization. This inference is substantiated by the chromophore chemical extraction data which show that, like the bovine system, octopus rhodopsin is an 11-cis pigment, while the photoproducts contain an all-trans pigment, in agreement with previous work. The octopus rhodopsin and bathorhodopsin spectra show marked differences from their bovine counterparts in other respects, however. The differences are most dramatic in the structure-sensitive fingerprint and the HOOP regions. Thus, it appears that although the two species differ in the specific nature of the chromophore-protein interactions, the general process of visual transduction is the same.     

Resonance Raman spectroscopy of carotenoids in Photosystem II core complexes

Resonance Raman (RR) spectroscopy has been used to examine the configuration of the carotenoids bound to Synechocystis PCC 6803 Photosystem II (PS II) core complexes. The excitation wavelengths used (514.5, 488.0, 476.5 and 457.9 nm) span the absorption bands of all of the ~12–17 neutral carotenoids in the PS II core complex. The RR spectra of the two carotenoids associated with the D1–D2 polypeptides (Car₅₀₇ and Car₄₈₉) of the reaction center are extracted via light versus dark difference experiments measured at 20 K. The RR results are consistent with all-trans configurations for both Car₅₀₇ and Car₄₈₉ and indicate that majority of the other carotenoids in the PS II core complex must also be in the all-trans configuration. The configuration of β-carotene is relevant to its proposed function as a molecular wire in the secondary electron-transfer reactions of PS II.     

Resonance Raman spectroscopy of sensory rhodopsin II from Natronobacterium pharaonis

Sensory rhodopsin II (pSRII), the photophobic receptor from Natronobacterium pharaonis, has been studied by time-resolved resonance Raman (RR) spectroscopy using the rotating cell technique. Upon excitation with low laser power, the RR spectra largely reflect the parent state pSRII(500) whereas an increase of the laser power leads to a substantial accumulation of long-lived intermediates contributing to the RR spectra. All RR spectra could consistently be analysed in terms of four component spectra which were assigned to the parent state pSRII(500) and the long-lived intermediates M(400), N(485) and O(535) based on the correlation between the C = C stretching frequency and the absorption maximum. The parent state and the intermediates N(485) and O(535) exhibit a protonated Schiff base. The C = N stretching frequencies and the H/D isotopic shifts indicate strong hydrogen bonding interactions of the Schiff base in pSRII(500) and O(535) whereas these interactions are most likely very weak in N(485).     

Sulfenylation of chiral Schiff bases

Summary The purpose of this study was to examine the conditions of sulfenylation reactions of chiral-amino esters Schiff bases to protect the chirality in the-position.     

Resonance Raman spectroscopy of xanthophylls in pigment mutant thylakoid membranes of pea

Low-temperature resonance Raman spectroscopy was used to study the changes in the molecular structure and configuration of the major xanthophylls in thylakoid membranes isolated from mutants of pea with modified pigment content and altered structural organization of their pigment-protein complexes. The Raman spectra contained four known groups of bands, nu(1)-nu(4), which could be assigned to originate mainly from the long wavelength absorbing lutein and neoxanthin upon 514.5 nm and at 488 nm excitations, respectively. The overall configuration of these bound xanthophyll molecules in the mutants appeared to be similar to the wild type, and the configuration in the wild type was almost identical with that in the isolated main chlorophyll a/b light harvesting protein complex of photosystem II (LHCII). Significant differences were found mainly in the region of nu(4) (around 960 cm(-1)), which suggest that the macroorganization of PS II-LHCII supercomplexes and/or of the LHCII-only domains are modified in the mutants compared to the wild type.     

Resonance Raman spectroscopy of methylamine dehydrogenase from bacterium W3A1

Resonance Raman spectroscopy has been used to probe the structure of the covalently bound quinone cofactor in methylamine dehydrogenase from the bacterium W3A1. Spectra were obtained on the phenylhydrazine and 2-pyridylhydrazine derivatives of the native enzyme, on the quinone-containing subunit labeled with phenylhydrazine, and on an active-site peptide also labeled with phenylhydrazine. Comparisons of these spectra to the corresponding spectra of copper-containing amine oxidase derivatives indicate that the quinones in these two classes of quinoproteins are not identical. The resonance Raman spectra of the native enzyme and small subunit have also been measured. 16O/18O exchange permitted the carbonyl modes of the quinone to be identified in the resonance Raman spectrum of oxidized methylamine dehydrogenase: a band at 1614 cm-1, together with a shoulder at 1630 cm-1, are assigned as modes containing substantial C = O stretching character. D2O/H2O exchange has pronounced effects on the resonance Raman spectrum of the oxidized enzyme, suggesting that the quinone may have numerous hydrogen bonds to the protein or that it is unusually sensitive to the local environment. Resonance Raman spectra of the isolated small subunit, and its phenylhydrazine derivative, are considerably different from the corresponding spectra of the intact protein. An attractive explanation for these observations is that the quinone cofactor in methylamine dehydrogenase from W3A1 is located at the interface between the large and small subunits, as found for the enzyme from Thiobacillus versutus [Vellieux, F. M. D., Huitema, F., Groendijk, H., Kalk, K. H., Frank, J. Jzn., Jongejan, J. A., & Duine, J. A. (1989) EMBO J. 8, 2171-2178].(ABSTRACT TRUNCATED AT 250 WORDS)