Marc Lutz

Structures of antenna complexes of several Rhodospirillales from their resonance Raman spectra

Several antenna complexes of Rhodospirillales were studied, within the intracytoplasmic membrane or in their isolated states by comparing resonance Raman spectra of their bacteriochlorophyll (BChl) molecules. It has been found that in B880-type complexes the states of BChl are extremely similar, in terms of their local environments. By contrast, at least two families of structures must be distinguished among the B850-800-type complexes, namely B850-800 S (from Chromatiaceae) and B850-800 NS (from Rhodospirillaceae). It appears that the more the energy of the lower singlet level of antenna BChl is decreased from its value in the isolated state, the smaller is the observed variability in its proteic host sites in the set of complexes observed. On the other hand, resonance Raman spectroscopy permits to conclude that the ground-state interactions assumed by the dihydrophorbin ring of BChl a within these complexes most probably are protein-BChl and not BChl-BChl or lipids-BChl interactions. Histidine is a likely candidate as the Mg ligand for the 880 and 850 nm absorbing molecules, but not for the 800 nm absorbing ones.

The stereoisomerism of bacterial, reaction-center-bound carotenoids revisited: An electronic absorption, resonance Raman and 1H-NMR study

Abstract In order to solve discrepancies between earlier assignments we have reinvestigated the stereoisomerism of the spheroidene molecule bound to reaction centers (RC) of Rhodobacter sphaeroides. A stable cis isomer could be extracted and purified from the reaction centres by working at very low ambient light. Resonance Raman spectroscopy showed that this cis isomer assumed the same configuration as that of the RC-bound molecule. Proton-NMR spectroscopy of the extracted isomer permitted to assign it the 15–15′ mono cis configuration. Comparisons between resonance Raman spectra of the native form and of the 15 cis extract showed that, in the reaction center, 15 cis spheroidene is in addition twisted into a non-planar conformation. Comparisons of extraction-induced changes in relative intensities of Raman bands of the 760–1060 cm−1 regions, which largely correspond to out-of-plane modes, further indicated that the out-of-plane twist of RC-bound spheroidene should predominantly affect C8–C12 and/or C8′–C12′ regions of the molecule rather than the central region. Comparisons between difference electronic absorption spectra of RC-bound spheroidene and of RC-bound methoxyneurosporene showed that the out-of-plane twisting of both these native forms results in a drastic weakening of their 1C ← 1A electronic transitions, compared with those of the planar, 15 cis forms. Finally, it is proposed, on the basis of their resonance Raman spectra, that spirilloxanthin bound to RCs of Rhodospirillum rubrum as well as dihydroneurosporene or dihydrolycopene bound to RCs of Rhodopseudomonas viridis shares 15 cis configurations and out-of-plane twisting with carotenoids bound to RCs of various strains of Rb. sphaeroides.

Selective photochemical reduction of either of the two bacteriopheophytins in reaction centers of Rps. sphaeroides R-26

The bacterial photosynthetic reaction center (RC) contains two bacteriopheophytin (BPh) molecules, only one of which (characterized by Qx,y maxima at 540 and 760 nm) has been found to function in the normal photochemistry. We present here the formation of a new RC redox state, in which the apparently inactive BPh (Qx,y maxima at 530 and 752 nm) is selectively trapped in a reduced state by secondary photochemistry. This BPh is found to be associated to a bacteriochlorophyll (BChI), forming a BChl‐BPh complex analogous to the photochemically active BChl‐BPh acceptor complex. Electron transfer between the two BPhs is found not to occur.

Bacteriochlorophyll a-protein interactions in a complex from Prosthecochloris aestuarii. A Resonance Raman study

Raman spectra of bacteriochlorophyll a (BChl) bound to the soluble protein complex from Prosthecochloris aestuarii have been obtained at low temperature, using the resonance effect on their Qx for Soret electronic bands. These spectra show that the acetyl carbonyls of at least four of the seven molecules bound to the monomer subunit of the complex and the ketone carbonyls of at least five of them are oriented close to the mean plane of the conjugated part of the dihydrophorbin macrocycle. Up to three bacteriochlorophyll molecules may have their ketone carbonyls free from hydrogen-bonding and up to two may have their acetyl carbonyls similarly free. Several of the binding sites of the remaining conjugated carbonyls are probably the same as those binding the conjugated carbonyls of bacteriochlorophyll (and of bacteriopheophytin) in reaction centers and in antenna structures of purple bacteria and as those binding chlorophyll in the antenna of higher plants and algae. The present resonance Raman spectra confirm that the magnesium atoms of most of the seven bacteriochlorophylls are pentacoordinated. They also show that polarisation effects from their local environments induce changes in the groundstate structures of the dihydrophorbin skeletons of the complexed molecules with respect to those of isolated, monomeric bacteriochlorophyll. These changes are quasi-identical for the seven molecules. These environmental effects predominate over any structural change brought about by intermolecular bonding of the conjugated carbonyls or of the magnesium atoms. The dihydrophorbin rings of the seven molecules thus appear to be immersed in a nearly homogeneous medium of low permittivity, although specific van der Waals interactions may polarise the free carbonyls to quite different extents. The possible implications of these observations on the interpretation of the electronic spectrum of the set of complexed bacteriochlorophylls are discussed.

Application of near-IR Fourier transform resonance Raman spectroscopy to the study of photosynthetic proteins

Abstract We demonstrate the application of near-infrared (NIR) Fourier transform (FT) Raman spectroscopy using 1064 nm excitation to obtain high quality preresonance Raman spectra of bacteriochlorophyll chromophores in photosynthetic proteins from purple bacteria at room temperature without sample degradation. We present NIR FT preresonance Raman spectra of chromatophores from Rhodospirillum (Rsp.) rubrum, carotenoidless strain G9 containing bacteriochlorophyll a (BChl a) chromophores mostly from its B880 antenna complex, and the B850-800 antenna complex from Rhodobacter (Rb.) sphaeroides, 2.4.1 strain; these spectra are compared with their resonance Raman (RR) spectra obtained using 363.8 nm excitation at 30 K. For antenna complexes not containing carotenoid the FT Raman spectra are dominated by the vibrational modes of the bacteriochlorophyll chromophores with no interference from the modes of the protein or membrane. In the NIR FT Raman spectrum of the B850-800 complex from Rb. sphaeroides 2.4.1, strong contributions from the carotenoid molecule are observed to cause some interference with the 1609 cm−1 band of the BChl a molecules. We also present FT Raman spectra of reaction centers (RCs) from Rb. sphaeroides R 26 in the reduced and oxidized states of their primary electron donor (P). In the reduced state, it is estimated that ca 70% of the FT Raman spectrum arises from reduced P whose electronic absorption band is at 865 nm. With 1064 nm excitation of the RCs poised in the oxidized cation radical state of P, we have observed for the first time a Raman spectrum of P+. via resonance in the vibronic region of the 1250 nm absorption band of this species. This spectrum indicates that the unpaired electron in dimeric P+. is not equally shared between the two BChl molecules constituting the primary donor. Spectral contributions of the carotenoid in the FT RR spectra of wild-type Rb. sphaeroides (2.4.1) RCs confirm it is assuming an out-of-plane distorted, 15-cis configuration under the specific conditions offered by FT Raman spectroscopy, i.e. with the sample at room temperature and using an excitation wavelength which is non-actinic for the carotenoid.

High-yield chemical assembly of [2Fe-2X] (X = S, Se) clusters into spinach apoferredoxin: product characterization by resonance Raman spectroscopy

Abstract Conditions are described for the non-enzymic assembly of [2Fe-2S] or [2Fe-2Se] chromophores into spinach apoferredoxin with high yields (60% and 40%, respectively) under physiologically relevant conditions. The success of these reconstitution reactions was found to be critically dependent on the conditions used for the denaturation of native ferredoxin (0.5 M HCl, anaerobic conditions). Low-temperature resonance Raman spectra of native and Se-substituted spinach ferredoxin have been recorded and compared. Most spectral features are shifted to lower frequencies upon S ∗ → Se ∗ substitution, due to the larger atomic mass of selenium compared to sulfur. As a result, each of the spectra displays characteristic bands which are absent in the other. This observation has been used to show that some preparations of Se-substituted ferredoxin also contain [2Fe-2S] chromophores, most probably arising from residual inorganic sulfur bound to apoprotein prepared under non-optimal conditions. Quantitative estimations have shown that the presence of a few percent of residual sulfur in Se-substituted ferredoxin can be detected by resonance Raman spectroscopy. This technique has in addition been used to demonstrate that ferredoxin preparations containing both chalcogenides involve hybrid [2Fe-S-Se] clusters in addition to the [2Fe-2S] and [2Fe-2Se] ones.

Resonance raman spectroscopy of protoheme-protein interactions in oxygen-carrying hemoproteins and in peroxidases

Abstract Resonance Raman spectra of deoxygenated ferrous forms of horse myoglobin, soybean leghemoglobin (isoenzyme a ), monomeric hemoglobin from Glycera, Aplysia myoglobin, horseradish peroxidase (isoenzyme C) and of turnip peroxidase (isoenzymes 1 and 7), excited at 441.6 nm, are reported. Differences observed among these spectra show that the proteins of these two classes of hemoprotein (oxygen-carriers and peroxidases) impose two distinct heme structures which are likely related to their biological functions. Raman bands involving stretching or deformation modes of the vinyl or propionyl groups of the porphyrin occur at different frequencies in the two classes of hemoprotein, reflecting protein-induced differences in conformations of these side-chains. Moreover, resonance Raman spectra allow a diagnosis of vinyl conformations in hemoproteins. More generally, differences in protein environments of the peripheral grous of protoheme in oxygen-carrying hemoproteins and in peroxidases explain the better electron-withdrawing capabilities of these groups in peroxidases than in oxygen-carriers. In addition, the low-frequency regions of resonance Raman spectra show that the axial histidylimidazole is probably deprotonated in the plant peroxidases, while it remains protonated for the oxygen-carriers. This spectroscopic approach, pointing to differences in the peripheral and axial parts of proteheme in the two classes of hemoprotein, provides a new understanding of structure-function relationships of protoheme in hemoproteins.

CHEMICALLY MODIFIED PHOTOSYNTHETIC BACTERIAL REACTION CENTERS: CIRCULAR DICHROISM, RAMAN RESONANCE, LOW TEMPERATURE ABSORPTION, FLUORESCENCE AND ODMR SPECTRA AND POLYPEPTIDE COMPOSITION OF BOROHYDRIDE TREATED REACTION CENTERS FROM Rhodobacter sphaeroides R26

Abstract— Reaction centers from Rhodobacter sphaeroides have been modified by treatment with sodium borohydride similar to the original procedure [Ditson et al., Biochim. Biophys. Acta766, 623 (1984)], and investigated spectroscopically and by gel electrophoresis.

Low-frequency vibrations of ferroprotoporphyrin-substituted imidazole complexes. A resonance Raman study

This article reports the low-frequency regions of resonance Raman spectra of five- and six-coordinated ferroprotoporphyrin complexes in aqueous solution with or without detergent. For high-spin complexes having their iron atom monocoordinated to variously substituted imidazoles or to dimethylformamide, the frequency of a band observed between 194 and 237 cm−1 (labelled band II) primarily depends on the pKa of the axial ligand. In the absence of steric effects from the axial ligand, the lower is the pKa of ligand, the higher the frequency of band II. We previously assigned band II to a mode essentially involving the Fe-N(pyrrole) bonds. The above pKa dependence is readily explained, in the frame of this assignment, in terms of a decrease in the Fe-N(pyrrole) bond strength (and of an increase in bond length) when the basicity of the axial ligand increases. On the other hand, the alternative assignment of band II to a stretching mode of Fe-N(axial ligand) is inconsistent with the observed pKa dependence. As far as hexacoordinated complexes are concerned, specific bands are observed at 203, 194 and 176 cm−1 for imidazole, 1-methylimidazole and pyridine, respectively. These bands are assigned, on the basis of isotopic substitutions, to a summetric stretching mode of the axial ligands [ν(N-Fe-N)]. Band II is observed at 265 cm−1 for these low-spin complexes, a frequency expected from the short Fe-N(pyrrole) bond lengths of nearly planar ferroporphyrins.

Replacement Of Sulfur By Selenium In Iron—Sulfur Proteins

Publisher Summary This chapter describes the replacement of sulfur by selenium in iron–sulfur proteins. By its chemical properties, selenium is most similar to sulfur and occurs in the same valence states: −2, 0, +2, +4, and +6. However, the two elements display noteworthy differences relevant to the biochemistry of selenium. Selenium tends to be more stable than sulfur in its intermediate oxidation states and less stable in the extreme ones. Accordingly, selenate and selenite are relatively easy to reduce to the element, and selenides are more reactive (reducing) than sulfides. Selenium is most often found in biological systems in compounds such as selenols, diselenides, and selenoethers, which are usually more reactive than their sulfur counterparts, because of the greater polarity and lower strength of the C–Se, N–Se and O–Se bonds. Selenols are more acidic (usually ionized at neutral pH), are better nucleophiles, better leaving groups, and are more reducing than the corresponding thiols.