Biomedical subjects

J Breton

Publications and source records attributed to J Breton.

At least 73 records · Page 4Linked to original sources

PubMed1995

Structural model of the photosynthetic reaction center of Rhodobacter capsulatus.

The reaction center (RC) from the photosynthetic bacterium Rhodobacter (Rb.) capsulatus has been the subject of a considerable amount of molecular biological and spectroscopic work aimed at improving our understanding of the primary steps of photosynthesis. However, no three-dimensional structure is available for this protein. We present here a model obtained by combining information from the structure of the highly homologous RC from Rhodopseudomonas (Rps.) viridis with molecular mechanics and simulated annealing calculations. In the Rb. capsulatus model the orientations of the bacteriochlorophyll monomer and the bacteriopheophytin on the branch inactive in electron transfer differ significantly from those in the RCs of Rps. viridis and Rb. sphaeroides. The bacteriopheophytin orientational difference is in good accord with previous linear dichroism measurements. A comparison is made of interactions between the pigments and the protein environment that may be of functional significance in Rps. viridis, Rb. sphaeroides, and Rb. capsulatus.

Amino Acids↗

PubMed1994

Coherent nuclear dynamics at room temperature in bacterial reaction centers.

A room-temperature study is reported of the femtosecond spectral evolution of the stimulated emission band of the primary electron-transfer precursor P* in bacterial photosynthesis. The study was performed with membranes of the antenna-deficient RCO1 mutant of Rhodobacter sphaeroides. A time-dependent red shift, reflecting nuclear motion out of the Franck-Condon region of the excited state, is resolved. Analysis of oscillatory features persisting for > 1 ps in the kinetics revealed main frequencies of the activated motions at 30, 84, 145, and 192 cm-1. The oscillations occur on the time scale of primary electron transfer. Our results set a lower limit for the vibrational dephasing time in P* that is not compatible with the usual assumption in theoretical treatments of complete vibrational relaxation prior to electron transfer, even at room temperature.

Bacteriochlorophylls↗

PubMed1994

Binding sites of quinones in photosynthetic bacterial reaction centers investigated by light-induced FTIR difference spectroscopy: assignment of the interactions of each carbonyl of QA in Rhodobacter sphaeroides using site-specific 13C-labeled ubiquinone.

Light-induced QA-/QA FTIR difference spectra of the photoreduction of the primary quinone (QA) have been obtained for Rhodobacter sphaeroides reaction centers (RCs) reconstituted with ubiquinone (Q3) labeled selectively with 13C at the 1- or 4-position of the quinone ring, i.e., on either of the two carbonyls. The vibrational modes of the quinone in the QA site are compared to those in vitro. IR absorption spectra of films of the labeled quinones show that the two carbonyls contribute equally to the split C = O band at 1663-1650 cm-1. This splitting is assigned to the two different geometries of the methoxy group nearest to each carbonyl. The QA-/QA spectra of RCs reconstituted with either 13C1- or 13C4-labeled Q3 and with unlabeled Q3 as well as the double differences calculated from these spectra exhibit distinct isotopic shifts for the bands assigned to C = O and C = C vibrations of the neutral QA. For the unlabeled QA, these bands correspond to the bands at 1660, 1628, and 1601 cm-1 previously detected upon nonselective isotopic labeling [Breton, J., Burie, J.-R., Berthomieu, C., Berger, G., & Nabedryk, E. (1994) Biochemistry 33, 4953-4965]. The 1660-cm-1 band is unaffected upon selective labeling at C4 but shifts to approximately 1623 cm-1 upon 13C1 labeling, demonstrating that this band arises from the C1 carbonyl, proximal to the isoprenoid chain. The band at 1628 cm-1 shifts by 11 and 16 cm-1 upon 13C1 and 13C4 labeling, respectively, and is assigned to a C = C mode coupled to both carbonyls.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbon Isotopes↗

PubMed1994

MCD and 1H-NMR spectroscopic studies of Desulfovibrio africanus ferredoxin I: revised amino-acid sequence and identification of secondary structure.

Desulfovibrio africanus ferredoxin I was studied by magnetic circular dichroism and 1H-NMR spectroscopies. These showed the presence of histidine and tryptophan, in contrast to the previously reported amino-acid sequence (Bruschi and Hatchikian (1982) Biochimie 64, 503-507). This was redetermined and the revised sequence shown to contain both histidine and tryptophan, as well as four other corrections (Sery et al. (1994) Biochemistry, submitted). Electrospray mass spectrometry confirmed the mass of the ferredoxin was that given by the revised amino-acid sequence. The secondary structure of the ferredoxin I was investigated with two-dimensional 1H-NMR experiments and both alpha-helix and beta-sheet structure detected. The influence of the paramagnetism of the Fe4 S4 cluster on the NMR properties of the ferredoxin protons was investigated, by temperature-dependent experiments, and it was concluded that there is only a negligible dipolar contribution to resonance chemical shifts from this source. The significance of this for the determination of the three-dimensional structure of the ferredoxin by NMR is discussed.

Amino Acid Sequence↗

PubMed1994

Binding sites of quinones in photosynthetic bacterial reaction centers investigated by light-induced FTIR difference spectroscopy: binding of chainless symmetrical quinones to the QA site of Rhodobacter sphaeroides.

Light-induced FTIR QA-/QA difference spectra corresponding to the photoreduction of the primary quinone acceptor QA have been obtained for Rhodobacter sphaeroides RCs reconstituted with chainless symmetrical quinones in order to study the influence of the side chain and of molecular asymmetry on the binding of natural quinones to the QA site. The main vibrational modes of the quinones in vivo were obtained by analysis of the isotope effects induced by 18O substitution on the carbonyls and by comparison with the IR absorption spectra of the isolated quinones. For isolated 2,3-dimethoxy-5,6-dimethyl-1,4-benzoquinone (MQ0), 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone, DQ), and 2,3-dimethyl-1,4-naphthoquinone (DMNQ), the IR spectra together with mass spectroscopy data of partially 18O labeled quinones show that the labeling of one carbonyl leads to only a minor shift of the vibrational frequency of the opposite carbonyl. This observation demonstrates an essentially uncoupled behavior of the two C = O groups. Upon reconstitution of QA-depleted RCs with these symmetrical quinones, the double-difference spectra calculated from the QA-/QA spectra of the 18O-labeled and unlabeled quinones reveal a splitting of the quinone C = O modes. This splitting and the frequency downshift of the C = O vibrations upon binding to the QA site are comparable to those previously reported for the C = O modes of quinones containing an isoprenoid (Q8, Q6, Q1) or a phytyl chain (vitamin K1) [Breton, J., Burie, J.-R., Berthomieu, C., Berger, G., & Nabedryk, E. (1994) Biochemistry 33, 4953-4965]. This observation demonstrates that the replacement of the side chain by a methyl group does not impair the asymmetrical bonding interactions of the two quinone carbonyls with the protein. This asymmetry is traceable to the two distinct amino acid residues which have been proposed, on the basis of X-ray structural studies, to form hydrogen bonds with the carbonyls of the quinone. The close analogy between the double-difference spectra calculated for RCs reconstituted either with vitamin K1 or with DMNQ shows that the phytyl chain of vitamin K1 imparts no specific constraint on the geometry of the menaquinone head group in its binding site for both the neutral and the semiquinone state. In contrast, the double-difference spectra calculated for RCs reconstituted either with MQ0 or with Q6 (or Q1) exhibited significant differences in the relative amplitudes of the bands assigned to the mixed C = O and C = C modes of the neutral quinones.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗

PubMed1994

Molecular cloning of rat kynurenine aminotransferase: identity with glutamine transaminase K.

The enzyme kynurenine aminotransferase (KAT) catalyses the conversion of L-kynurenine to kynurenic acid. A combination of polymerase chain reaction techniques and hybridization screening was used to isolate a cDNA clone encompassing the entire coding region of KAT from rat kidney. Identification of the cDNA as coding for KAT was based both on the comparison of amino acid sequences obtained from purified rat KAT and on the expression of KAT activity in COS-1 cells transfected with the cDNA. RNA blot analysis indicated that KAT mRNA is widely expressed in rat tissues. Cultured cells transfected with the cDNA for KAT also showed glutamine transaminase K activity. Based mainly on sequence data, these results demonstrate that rat kidney KAT is identical with glutamine transaminase K.

Amino Acid Sequence↗

PubMed1994

Gaussian decomposition of absorption and linear dichroism spectra of outer antenna complexes of photosystem II.

Room temperature and 10 K absorption and linear dichroism spectra of the chlorophyll-protein complexes comprising the outer antenna of PSII (LHCII, CP29, CP26, CP24) have been analyzed in terms of a linear combination of asymmetric Gaussian bands. The results demonstrate the following: (a) The absorption and linear dichroism spectra of each sample can be described by nearly the same set of Gaussian bands at room temperature and 10 K. (b) The relative distributions of the transition moments of the major red-absorbing spectral forms seem to be similar in all four outer antenna chlorophyll-protein complexes at room temperature, with the 684-nm band being oriented closest to the particle plane at room temperature and the 677- and 669-nm bands being tilted at progressively greater angles out of the particle plane. The shorter wavelength transitions seem to be oriented close to the magic angle, but interpretation is complicated in this spectral region due to the low linear dichroism values and by overlap with vibrational bands. (c) The 684-nm band, detected in room temperature absorption and linear dichroism spectra of all complexes, vanishes at 10 K.

Chlorophyll↗

PubMed1994

Primary charge separation in photosystem I: a two-step electrogenic charge separation connected with P700+A0- and P700+A1- formation.

A PS I membrane preparation from a PS II deficient mutant of Synechocystis sp. PCC 6803 (psb DI/DII/C) was investigated by picosecond photovoltage and fluorescence measurements. The photovoltage kinetics show two distinct phases. At low excitation energies the fast phase correlates with the fluorescence decay time constant of 22 +/- 4 ps. This phase is ascribed to the trapping of excitons as described by the reaction (AntiP700)* A0-->AntiP700+A0-. In addition to this phase, the photovoltage displays a second rising phase of smaller amplitude with a time constant of 50 +/- 15 ps. We assign the latter phase to further electron transfer from A0 to the secondary acceptor, A1. Assuming the protein as a homogeneous dielectric, our results suggest that the transmembrane distance A0-A1 spans only a small part (20 +/- 8%) of the distance P700-A1.

Chlorophyll↗

PubMed1994

Coherent dynamics during the primary electron-transfer reaction in membrane-bound reaction centers of Rhodobacter sphaeroides.

The temporal evolution of the near-infrared stimulated emission band of the special pair excited state (P*) in the reaction center of Rhodobacter sphaeroides has been studied in intracytoplasmic membranes of the antenna-deficient RCO1 mutant at 10 K with a resolution of 30 fs. On the 100-fs time scale the emission band gradually shifts to longer wavelengths. After 150 fs the band shifts back to shorter wavelengths and continues to develop on the picosecond time scale in a damped oscillatory manner (most prominent fundamental frequencies around 15 cm-1 and at 92, 122, and 153 cm-1). These phenomena are shown to be due to low-frequency vibrational motions in the P* excited state that conserve their phase on the time scale of electron transfer. These results imply that the vibrational manifold of P* is not thermalized during the electron-transfer reaction in functional reaction centers. The initial Stokes shift dynamics are largely determined by the modes in the 90-160-cm-1 frequency range, which probably involve motions of several chromophores, including the bacteriopheophytin electron acceptor HL.

Electron Transport↗

PubMed1994

Characterization of the paramagnetic iron-containing redox centres of Thiosphaera pantotropha periplasmic nitrate reductase.

Electron paramagnetic resonance spectroscopy signals attributable to low-spin haem c in the oxidised protein and [4Fe-4S]1+ in the dithionite-reduced protein were identified, at low temperature, in Thiosphaera pantotropha periplasmic nitrate reductase. Spin integration of these signals as well as elemental analysis suggest a stoichiometry of 1.3-1.6 c-haem and 1 [4Fe-4S] cluster per enzyme molecule. The Em (at pH 7.4) of the [4Fe-4S]2+,1+ couple, -160 mV, means that it is unlikely to be physiologically reducible. Peptide sequences from the 90 kDa subunit indicate that the enzyme is a member of the family of molybdopterin guanine dinucleotide-binding polypeptides, the majority of which possess a putative [4Fe-4S] cluster binding sequence and thus may also bind a (low potential) iron-sulphur cluster.

Amino Acid Sequence↗

PubMed1994

The binding sites of quinones in photosynthetic bacterial reaction centers investigated by light-induced FTIR difference spectroscopy: assignment of the QA vibrations in Rhodobacter sphaeroides using 18O- or 13C-labeled ubiquinone and vitamin K1.

Light-induced FTIR difference spectra of the photoreduction of the primary quinone acceptor QA have been obtained for Rhodobacter sphaeroides RCs reconstituted with a series of isotopically labeled quinones in order to separate the contributions of the quinone from those of the protein. The isotopic shifts observed in the QA-/QA spectra of RCs reconstituted with ubiquinones (Q1, Q6) or vitamin K1 18O-labeled on their carbonyl oxygens and with fully 13C-labeled Q8 lead to a clear identification of the quinone bands from both the neutral and anion forms. Double-difference spectra from pairs of QA-/QA spectra obtained from 18O/16O Q6, 18O/16O Q1, 13C/12C Q8, 13C18O/12C16O Q8, and 18O/16O vitamin K1 allow the C = O modes of QA in vivo to be identified unambiguously for the first time. For all the investigated unlabeled quinones, two carbonyl bands are demasked, at 1660 and 1628 cm-1 for neutral ubiquinones and at 1651 and 1640 cm-1 for vitamin K1, while C = C bands are found at 1608 and 1588 cm-1 for vitamin K1 and at 1601 cm-1 for ubiquinones. Compared with the spectra of the isolated quinones, the generally smaller width observed for the C = O and C = C bands in vivo suggests precise interactions between the quinone and the contours of the protein at a single, well-defined QA site. The different frequency downshifts of the two C = O bands upon binding to the QA site underscore the inequivalence of the two carbonyls in providing asymmetrical bonding interactions with the protein. The comparison of the isotopic shifts observed for the various quinone C = O and C = C bands in vitro and in vivo demonstrates that the admixture of C = O and C = C characters in these modes is strongly affected by the binding of QA to its anchoring site. In particular, the bands at 1628 and 1601 cm-1 of Q6 in vivo exhibit highly mixed C = O and C = C characters. In contrast, the methoxy groups of the ubiquinones do not appear to suffer large strain upon binding. The closeness of the QA-/QA spectra for Q1 and Q6 indicates that a possible role of the chain in providing the proper positioning of the quinone ring in the site for both the oxidized and reduced states of QA cannot extend significantly beyond the first isoprene unit.(ABSTRACT TRUNCATED AT 400 WORDS)

Binding Sites↗

PubMed1994

Cysteine-rich LIM domains of LIM-homeodomain and LIM-only proteins contain zinc but not iron.

The structure of LIM domains has major implications for transcription because proteins such as Is1-1 contain two LIM domains associated with a homeodomain, and RBTN1/Ttg-1 and RBTN2/Ttg-2 contain two LIM domains but no homeodomain. Conserved cysteine and histidine residues in the LIM domains suggest a metal-binding role. RBTN and Is1-1 LIM proteins have been made in Escherichia coli and insect cell expression systems and their metal content has been determined using atomic absorption spectroscopy and electron paramagnetic resonance spectroscopy. LIM proteins expressed in soluble form contain zinc atoms, whereas bacterial inclusion bodies invariably also have Fe-S clusters. The latter are identified as linear [Fe3S4]+ clusters and appear to result from incorrect metal coordination by E. coli. These studies show that RBTN1, RBTN2, and Is1-1 are metalloproteins that contain zinc but not iron and, therefore, that the LIM domain represents a zinc-binding domain.

Cysteine↗

PubMed1994

Structure and function of the tetraheme cytochrome associated to the reaction center of Roseobacter denitrificans.

We have characterized the tetrahemic RC bound cytochrome isolated from the quasi-photosynthetic bacterium Roseobacter denitrificans in terms of absorption spectrum, redox property and orientation with respect to the membrane plane. The heme, designated H1, which possesses the highest redox midpoint potential (+290 mV), absorbs at 555 nm. Its plane makes an angle of 40 degrees with the membrane plane. The second high potential heme, H2 (+240 mV), peaks at 554 nm and makes a tilt of 55 degrees with the membrane. The two low potential hemes, L1 and L2, present a similar and rather high redox midpoint potential (+90 mV). They absorb at 553 nm and 550 nm. One of these hemes is oriented at 40 degrees while the other makes an angle of 90 degrees with the membrane plane. The soluble cytochrome c551 completes the cyclic electron transfer between the RC and the bc1 complex. Both the oxidation and the re-reduction of cytochrome c551 are diffusible processes. Under semi-aerobic conditions, one of the low potential hemes is photo-oxidized under illumination but only extremely slowly re-reduced. This explains the requirement of high aerobic conditions for growth of Roseobacter denitrificans cells in the light.

Bacteria↗

PubMed1994

Formation and properties of a stable 'high-potential' copper-iron-sulphur cluster in a ferredoxin.

A ferredoxin isolated from Desulfovibrio africanus contains a [3Fe-4S] cluster that reversibly binds a copper atom, yielding a stable product with a greatly increased reduction potential. The reaction is readily detected in protein molecules adsorbed as a film on an electrode surface. Electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectra of oxidized and reduced bulk solution products support their assignment as [Cu3Fe-4S]2+ (S = 1/2) and [Cu3Fe-4S]1+ (S = 2) respectively, with copper bound formally as Cu(I). Cyanide causes selective loss of copper and regeneration of the [3Fe-4S] reactant. The results demonstrate the chemical feasibility of CuFeS clusters and suggest that they could exist naturally in biological systems.

Amino Acid Sequence↗

PubMed1994

Human keratinocytes possess an sn-2 acylhydrolase that is biochemically similar to the U937-derived 85-kDa phospholipase A2.

The phospholipase A2 (PLA2) activities that are localized in the keratinocyte cytosolic and microsomal fractions were biochemically and pharmacologically characterized. The cytosol and to a lesser extent the microsome were sensitive to heat treatment and stable in the presence of sulfhydryl reducing agents. Both fractions were almost totally inactivated by reduction of pH to 2. The cytosolic activity demonstrated a sevenfold preference for arachidonic acid over oleic acid in the sn-2 position of substrate phospholipid and the microsome exhibited a fourfold preference. Neither the cytosol nor the microsome was inactivated by a neutralizing mouse monoclonal antibody 3F10 generated against recombinant human (rh) type II 14-kDa PLA2. Western immunoblot analysis of both fractions identified a high-molecular-mass protein in keratinocyte cytosol but not the microsome that migrated with rh 85-kDa PLA2. Neither the cytosol nor the microsome possessed immunoreactive bands that migrated with rh type II 14-kDa PLA2 when probed with monoclonal antibody 3F10. Further analysis of the cytosolic activity showed that it was activated by submicromolar concentrations of Ca2+, reduced by arachidonyl trifloromethylketone, a selective 85-kDa PLA2 inhibitor, but was unaffected by C-7 phosphonate phospholipid, a selective 14-kDa PLA2 transition state inhibitor. Taken together, the data supports the existence of a PLA2 activity in the cytosol that displays characteristics that are indistinguishable from those exhibited by the 85-kDa PLA2. Alternatively, both the cytosol and microsome were devoid of type II 14-kDa-like PLA2 activity. The failure of 12-epi scalaradial, a 14-kDa PLA2 inhibitor, to modify A23187-stimulated keratinocyte prostaglandin E2 release, was consistent with the biochemistry and suggests that the 85-kDa PLA2 may play an important role in keratinocyte prostaglandin E2 formation.

Antibodies, Monoclonal↗

PubMed1994

Interleukin-8 production is regulated by protein kinase C in human keratinocytes.

Interleukin-8 (IL-8) is a potent pro-inflammatory molecule present in high amounts in psoriatic skin. Here it may play an important role in the keratinocyte hyperproliferation and the neutrophil and T-lymphocyte infiltration associated with the disease. In this study the effect of protein kinase C inhibitors on IL-8 production by human keratinocytes in vitro was investigated. The anti-inflammatory and immunomodulatory compound auranofin ([1-thio-beta-D-glucopyranose-2,3,4,6-tetraacetato-S] [triethylphosphine] gold) is known to inhibit protein kinase C. In addition, auranofin has been shown to inhibit skin inflammation. As such, auranofin was also studied for its effect on IL-8 production. Auranofin and staurosporine, inhibitors of protein kinase C, inhibited phorbol-myristate-acetate-stimulated IL-8 production. Northern analysis of IL-8 mRNA revealed that the inhibition of IL-8 production was associated with an inhibition of IL-8 mRNA expression. In contrast, these compounds potentiated the minimal IL-8 protein and mRNA seen in response to interleukin-1 beta or tumor necrosis factor-alpha. These findings suggest that IL-8 synthesis may be either positively or negatively regulated by protein kinase C depending on the stimulus.

Adjuvants, Immunologic↗

PubMed1993

Fourier transform infrared study of the primary electron donor in chromatophores of Rhodobacter sphaeroides with reaction centers genetically modified at residues M160 and L131.

Structural changes in chromatophores of Rhodobacter sphaeroides reaction center mutants associated with the substitution of amino acid residues near the primary electron donor P have been investigated by light-induced FTIR difference spectroscopy. The single-site mutations Leu-L131 to His and Leu-M160 to His and the corresponding double mutation were designed to introduce a proton-donating residue that could form a hydrogen bond with the keto carbonyl of ring V of each bacteriochlorophyll (PL and PM) of the dimer. The presence of large positive bands at approximately 1550, 1480, and 1295 cm-1, as well as at 2600-2800 cm-1 in the light-induced P+QA-/PQA FTIR difference spectra, corresponding to the photooxidation of P and the photoreduction of the primary quinone QA, demonstrates that the BChl dimer state of P+ is preserved in the LH(L131), LH(M160), and LH(M160)+LH(L131) mutants, although frequency shifts and amplitude changes can be observed, notably for LH(M160). Compared to wild type, these changes are thought to reflect a different charge repartition over the two BChls in P+. Large frequency downshifts in the 9-keto C=O stretching region of the P+QA-/PQA FTIR difference spectra of chromatophores are observed in the mutant samples relative to wild type. For the LH(M160) mutant, a large differential signal at 1678/1664 cm-1 is assigned to a shift, upon photooxidation, of the 9-keto C=O of PM hydrogen-bonded to His-M160, while that at 1718/1696 cm-1 corresponds to the free 9-keto C=O of PL.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Chromatophores↗

PubMed1993

Evidence that serine L223 is involved in the proton transfer pathway to QB in the photosynthetic reaction center of Rhodopseudomonas viridis.

In the reaction center of purple photosynthetic bacteria, the reducing equivalents produced by primary charge separation are exported via an ubiquinone molecule working as a two-electron shuttle. This loosely-bound quinone, called QB, accepts in successive flashes two electrons from the tightly bound primary quinone acceptor QA, along with two protons from the external medium. The surrounding protein plays an important role in stabilizing the semiquinone anion and in providing a pathway for protons from the cytoplasmic phase to QB. Herbicides of the triazine type compete with QB for the binding pocket and their binding is controlled by nearby amino acid residues. We have studied the kinetics of the first and second electron transfer from QA to QB in two herbicide-resistant mutants from Rhodopseudomonas viridis, T1 (ArgL217-->His,Ser L223-->Ala) and MAV5 (Arg L217-->His, Val L220-->Leu), in order to determine whether these residues are involved in proton transfer to the reduced QB. The main effect of the mutant T1 was a drastic (600-fold at pH 7) decrease in the rate of the second electron transfer to QB compared to the wild type. In contrast, the rate of the second electron transfer in the mutant MAV5 was decreased only slightly (10-fold) in the pH range from 7 to 11. We attribute the inhibition of the second electron transfer in the Ser L223-->Ala mutation to an essential role of Ser L223 in the donation of the first proton to the reduced QB.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗