Biomedical subjects

J Breton

Publications and source records attributed to J Breton.

At least 37 records · Page 2Linked to original sources

PubMed1999

Orientation of chlorophyll transition moments in the higher-plant light-harvesting complex CP29.

The Q(y) transition dipole moment vectors of all eight chlorophylls in the higher-plant antenna protein CP29 were calculated by an original method on the basis of linear dichroism and absorption spectroscopy. The contribution of individual chromophores was determined from difference spectra between wild type and mutant proteins in which a single chlorophyll has been removed by mutating pigment-binding residues. Recombinant proteins were constructed by overexpressing the apoprotein in bacteria and refolding of the pigment-protein complex in vitro [Bassi, R., Croce, R., Cugini, D., and Sandonà, D. (1999) Proc. Natl. Acad. Sci. U.S.A. (in press)]. The spectroscopic data are interpreted on the basis of a protein structural model obtained via the homology with the major antenna complex LHCII [Kuhlbrandt, W., Wang, D. N., and Fujiyoshi, Y. (1994) Nature 367, 614-621]. The results allow us to determine the orientation of six chromophores within the protein structure. The orientations of the two remaining chromophores are inferred by considering the symmetry properties of CP29 and fitting steady state absorption and linear dichroism spectra by independent chlorophyll spectral forms. As a consequence, four "mixed" sites with different chlorophyll a and b binding affinities are identified in CP29. Geometrical data and the Förster mechanism for energy transfer suggest that excitation energy equilibrates rapidly among chlorophyll "pure" sites while energy preferentially flows outward from chlorophyll "mixed" sites. The orientation of the dipole moments of two chlorophyll molecules symmetrically located at the center of the protein and parallel to the carotenoid transition vectors suggests a role in energy transfer from xanthophyll to chlorophyll.

Amino Acid Sequence↗

PubMed1999

FTIR study of the primary electron donor of photosystem I (P700) revealing delocalization of the charge in P700(+) and localization of the triplet character in (3)P700.

The effect of global (15)N or (2)H labeling on the light-induced P700(+)/P700 FTIR difference spectra has been investigated in photosystem I samples from Synechocystis at 90 K. The small isotope-induced frequency shifts of the carbonyl modes observed in the P700(+)/P700 spectra are compared to those of isolated chlorophyll a. This comparison shows that bands at 1749 and 1733 cm(-)(1) and at 1697 and 1637 cm(-)(1), which upshift upon formation of P700(+), are candidates for the 10a-ester and 9-keto C=O groups of P700, respectively. A broad and relatively weak band peaking at 3300 cm(-)(1), which does not shift upon global labeling or (1)H-(2)H exchange, is ascribed to an electronic transition of P700(+), indicating that at least two chlorophyll a molecules (denoted P(1) and P(2)) participate in P700(+). Comparisons of the (3)P700/P700 FTIR difference spectrum at 90 K with spectra of triplet formation in isolated chlorophyll a or in RCs from photosystem II or purple bacteria identify the bands at 1733 and 1637 cm(-)(1), which downshift upon formation of (3)P700, as the 10a-ester and 9-keto C=O modes, respectively, of the half of P700 that bears the triplet (P(1)). Thus, while the P(2) carbonyls are free from interaction, both the 10a-ester and the 9-keto C=O of P(1) are hydrogen bonded and the latter group is drastically perturbed compared to chlorophyll a in solution. The Mg atoms of P(1) and P(2) appear to be five-coordinated. No localization of the triplet on the P(2) half of P700 is observed in the temperature range of 90-200 K. Upon P700 photooxidation, the 9-keto C=O bands of P(1) and P(2) upshift by almost the same amount, giving rise to the 1656(+)/1637(-) and 1717(+)/1697(-) cm(-)(1) differential signals, respectively. The relative amplitudes of these differential signals, as well as of those of the 10a-ester C=O modes, appear to be slightly dependent on sample orientation and temperature and on the organism used to generate the P700(+)/P700 spectrum. If it is assumed that the charge density on ring V of chlorophyll a, as measured by the perturbation of the 10a-ester or 9-keto C=O IR vibrations, mainly reflects the spin density on the two halves of the oxidized P700 special pair, a charge distribution ranging from 1:1 to 2:1 (in favor of P(2)) is deduced from the measurements presented here. The extreme downshift of the 9-keto C=O group of P(1), indicative of an unusually strong hydrogen bond, is discussed in relation with the models previously proposed for the PSI special pair.

Cyanobacteria↗

PubMed1999

Conformational heterogeneity of the bacteriopheophytin electron acceptor HA in reaction centers from Rhodopseudomonas viridis revealed by Fourier transform infrared spectroscopy and site-directed mutagenesis.

The light-induced Fourier transform infrared (FTIR) difference spectra corresponding to the photoreduction of either the HA bacteriopheophytin electron acceptor (HA-/HA spectrum) or the QA primary quinone (QA-/QA spectrum) in photosynthetic reaction centers (RCs) of Rhodopseudomonas viridis are reported. These spectra have been compared for wild-type (WT) RCs and for two site-directed mutants in which the proposed interactions between the carbonyls on ring V of HA and the RC protein have been altered. In the mutant EQ(L104), the putative hydrogen bond between the protein and the 9-keto C=O of HA should be affected by changing Glu L104 to a Gln. In the mutant WF(M250), the van der Waals interactions between Trp M250 and the 10a-ester C=O of HA should be modified. The characteristic effects of both mutations on the FTIR spectra support the proposed interactions and allow the IR modes of the 9-keto and 10a-ester C=O of HA and HA- to be assigned. Comparison of the HA-/HA and QA-/QA spectra leads us to conclude that the QA-/QA IR signals in the spectral range above 1700 cm-1 are largely dominated by contributions from the electrostatic response of the 10a-ester C=O mode of HA upon QA photoreduction. A heterogeneity in the conformation of the 10a-ester C=O mode of HA in WT RCs, leading to three distinct populations of HA, appears to be related to differences in the hydrogen-bonding interactions between the carbonyls of ring V of HA and the RC protein. The possibility that this structural heterogeneity is related to the observed multiexponential kinetics of electron transfer and the implications for primary processes are discussed. The effect of 1H/2H exchange on the QA-/QA spectra of the WT and mutant RCs shows that neither Glu L104 nor any other exchangeable carboxylic residue changes appreciably its protonation state upon QA reduction.

Amino Acid Substitution↗

PubMed1999

The neoxanthin binding site of the major light harvesting complex (LHCII) from higher plants.

The localisation of the xanthophyll neoxanthin within the structure of the major light harvesting complex (LHCII) of higher plants has been investigated by site-directed mutagenesis and spectroscopic methods. Mutation analysis performed on pigment binding sites in different helix domains leads to selective loss of neoxanthin for mutations on helix C thus localising this pigment between the helix C and helix A/B domains. Recombinant proteins binding two lutein molecules per polypeptide but lacking neoxanthin have been used in order to determine the contribution of neoxanthin to the absorption and linear dichroism spectra. The data were used to derive the orientation of the neoxanthin transition moment, lying in the polyene chain, which was thus determined to form an angle of 57 +/- 1.5 degrees with respect to the normal to the membrane plane where the protein is inserted. On the basis of these results we propose a model for the localisation of the carotenoid site in the LHCII structure which is still unresolved.

Binding Sites↗

PubMed1999

The VUV Absorption Spectra of SiF and SiF+

The absorption spectrum of a microwave discharge (2450 MHz) in flowing helium with traces of SiF4 has been recorded on the SA61 beam line of SUPERACO between 2000 and 500 Å. Two new ionization energies have been observed at 15.12 and 17.27 eV corresponding, respectively, to the A1Pi and B1Sigma+ states of SiF+. The A1Pi <-- X1Sigma+ transition of the ion has been identified for the first time on high-resolution spectra obtained in 1992 with the 35-foot Eagle spectrograph in Meudon. Copyright 1999 Academic Press.

Journal Article↗

PubMed1999

Rydberg States of SiCl.

A fluorescence spectrum, excited by the synchrotron radiation of Superaco in a microwave discharge through He + SiCl(4), leads to the observation of five new electronic states of SiCl, converging to the ground X(1)Sigma(+) state of the SiCl(+) ion. The (n s varsigma) and (n p pi) Rydberg series are extended respectively to n = 6 and n = 7. A new ionization limit is observed in absorption at 11.238 eV corresponding to the a(3)Pi electronic state of SiCl(+). Four new states are observed which converge to that limit. Copyright 1999 Academic Press.

Journal Article↗

PubMed1999

The VUV Absorption Spectrum of the GeO Molecule.

The absorption spectrum of GeO, between 2000 and 300 Å, was recorded using the facilities of the SUPERACO synchrotron radiation in Orsay, France. The first three ionization limits, corresponding to the X(2)Sigma(+), A(2)Pi, and B(2)Sigma(+) states of the GeO(+) ion, were confirmed at 11.2814, 11.42, and 15.048 eV, respectively. The fourth ionization limit corresponding to the C(2)Sigma(+) ionic state was measured for the first time at 31.47 eV, in reasonable agreement with theoretical calculations. Copyright 1999 Academic Press.

Journal Article↗

PubMed1998

Proton uptake by carboxylic acid groups upon photoreduction of the secondary quinone (QB) in bacterial reaction centers from Rhodobacter sphaeroides: FTIR studies on the effects of replacing Glu H173.

In the photosynthetic reaction center (RC) from Rhodobacter sphaeroides, Glu H173, located approximately 7 A from the center of the secondary quinone acceptor QB, is expected to contribute to proton uptake upon QB- formation in response to the movement of an electron in its vicinity. Steady-state FTIR difference spectroscopy provides a method to monitor proton uptake by carboxylic acids upon photochemical changes. The FTIR spectra corresponding to the photoreduction of QB were obtained at pH 7 for RCs containing Glu (native), Gln (EQ H173), or Asp (ED H173) at the H173 site. No new bands were observed in the carboxylic acid region (1770-1700 cm-1) in any of the mutant RCs compared to native RCs. In addition, the positive band at 1728 cm-1, previously assigned to Glu L212 [Nabedryk, E., Breton, J., Hienerwadel, R., Fogel, C., Mäntele, W., Paddock, M. L., and Okamura, M. Y. (1995) Biochemistry 34, 14722-14732], remained present in all of the mutant RCs. This result shows that Glu H173 is not a major contributor to proton uptake upon QB- formation and further strengthens the assignment of the 1728 cm-1 band to Glu L212. An increase in the 1728 cm-1 band was observed in the EQ H173 RCs compared to that of either the ED H173 or native RCs. These changes are consistent with Glu and Asp at H173 remaining ionized in the QB and QB- states. Changes in the absorption regions of the semiquinone and amide or side chain groups in the spectra of the mutant RCs suggest slight changes in the protein structure compared to those of native RCs, which could contribute to the altered kinetics observed in the mutant RCs.

Amino Acid Substitution↗

PubMed1998

Low frequency vibrational modes in proteins: changes induced by point-mutations in the protein-cofactor matrix of bacterial reaction centers.

As a step toward understanding their functional role, the low frequency vibrational motions (<300 cm-1) that are coupled to optical excitation of the primary donor bacteriochlorophyll cofactors in the reaction center from Rhodobacter sphaeroides were investigated. The pattern of hydrogen-bonding interaction between these bacteriochlorophylls and the surrounding protein was altered in several ways by mutation of single amino acids. The spectrum of low frequency vibrational modes identified by femtosecond coherence spectroscopy varied strongly between the different reaction center complexes, including between different mutants where the pattern of hydrogen bonds was the same. It is argued that these variations are primarily due to changes in the nature of the individual modes, rather than to changes in the charge distribution in the electronic states involved in the optical excitation. Pronounced effects of point mutations on the low frequency vibrational modes active in a protein-cofactor system have not been reported previously. The changes in frequency observed indicate a strong involvement of the protein in these nuclear motions and demonstrate that the protein matrix can increase or decrease the fluctuations of the cofactor along specific directions.

Bacterial Proteins↗

PubMed1998

Hydrogen bonding of redox-active tyrosine Z of photosystem II probed by FTIR difference spectroscopy.

The TyrZ./TyrZ FTIR difference spectrum is reported for the first time in Mn-depleted photosystem II (PS II)-enriched membranes of spinach, in PS II core complexes of Synechocystis sp. PCC 6803 WT, and in the mutant lacking TyrD (D2-Tyr160Phe). In Synechocystis, the v7'a(CO) and delta(COH) infrared modes of TyrZ are proposed to account at 1279 and 1255 cm-1. The frequency of these modes indicate that TyrZ is protonated at pH 6 and involved in a strong hydrogen bond to the side chain of a histidine, probably D1-His190. A positive signal at 1512 cm-1 is assigned to the v(CO) mode of TyrZ. on the basis of the 27 cm-1 downshift observed upon 13C-Tyr labeling at the Tyr ring C4 carbon. A second IR signal, at 1532 cm-1, is tentatively assigned to the v8a(CC) mode of TyrZ.. The frequency of the v(CO) mode of TyrZ. at 1512 cm-1 is comparable to that observed at 1513 cm-1 for the Tyr. obtained by UV photochemistry of tyrosinate in solution, while it is higher than that of TyrD. in WT PS II at 1503 cm-1 and that of non-hydrogen-bonded TyrD. in the D2-His189Gln mutant at 1497 cm-1 [Hienerwadel, R., Boussac, A., Breton, J., Diner, B. A., and Berthomieu, C. (1997) Biochemistry 36, 14712-14723]. This latter work and the present FTIR study suggest that hydrogen bonding induces an upshift of the v(CO) IR mode of tyrosyl radicals and that TyrZ. forms (a) stronger hydrogen bond(s) than TyrD. in WT PS II. Alternatively, the frequency difference between TyrZ. and TyrD. v(CO) modes could be explained by a more localized positive charge near the tyrosyl radical oxygen of TyrD. than TyrZ.. The TyrZ./TyrZ spectrum obtained in Mn-depleted PS II membranes of spinach shows large similarities with the S3'/S2' spectrum characteristic of radical formation in Mn-containing but Ca(2+)-depleted PS II, in support of the assignment using ESEEM of TyrZ. as being responsible for the split EPR signal observed upon illumination in these conditions [Tang, X.-S., Randall, D. W., Force, D. A., Diner, B. A., and Britt, R. D. (1996) J. Am. Chem. Soc. 118, 7638-7639]. The peak at 1514 cm-1 is assigned to the v(CO) mode of TyrZ. in these preparations, which indicates that Mn depletion only very slightly perturbs the immediate environment of TyrZ. phenoxyl.

Amino Acid Substitution↗

PubMed1998

A systematic survey of conserved histidines in the core subunits of Photosystem I by site-directed mutagenesis reveals the likely axial ligands of P700.

The Photosystem I complex catalyses the transfer of an electron from lumenal plastocyanin to stromal ferredoxin, using the energy of an absorbed photon. The initial photochemical event is the transfer of an electron from the excited state of P700, a pair of chlorophylls, to a monomer chlorophyll serving as the primary electron acceptor. We have performed a systematic survey of conserved histidines in the last six transmembrane segments of the related polytopic membrane proteins PsaA and PsaB in the green alga Chlamydomonas reinhardtii. These histidines, which are present in analogous positions in both proteins, were changed to glutamine or leucine by site-directed mutagenesis. Double mutants in which both histidines had been changed to glutamine were screened for changes in the characteristics of P700 using electron paramagnetic resonance, Fourier transform infrared and visible spectroscopy. Only mutations in the histidines of helix 10 (PsaA-His676 and PsaB-His656) resulted in changes in spectroscopic properties of P700, leading us to conclude that these histidines are most likely the axial ligands to the P700 chlorophylls.

Animals↗

PubMed1998

The VUV Absorption Spectrum of the Tin Monoxide Molecule

The absorption spectrum of heated SnO between 2000 and 300 A has been recorded using the facilities of the SuperACO synchrotron at Orsay, France. New bands have been observed which supplement the data on the previously known systems of SnO. Newly discovered Rydberg series have confirmed the four ionization potentials observed or calculated by Dyke et al. (1982. Chem. Phys. 67, 245). Copyright 1998 Academic Press. Copyright 1998Academic Press

Journal Article↗

PubMed1998

Evaluation of the cutaneous anti-inflammatory activity of azaspiranes.

OBJECTIVE AND DESIGN: The ability of azaspiranes to modulate the acute inflammatory response in models of skin inflammation was examined. MATERIAL: The in vivo experiments involved the use of 5-6 age-matched male Balb/c inbred mice (22-25 g) per treatment group and a control group of 8-10 animals. In vitro mechanistic studies used RBL-1 and U937 cells lines and freshly isolated human monocytes. TREATMENT: Arachidonic acid (AA) (2 mg/20 microl in acetone) or PMA (phorbol myristate acetate) (4 microg/20 microl) were applied topically. SK&F 106615 and SK&F 106610 were administered topically either dissolved in acetone or dimethylacetamide just after the application of the irritant. Isolated cells were treated with the compounds dissolved in DMSO. METHODS: The thickness and influx of neutrophils into the treated ears was measured as was the effects of the azaspiranes on 5-lipoxygenase activity, cyclooxygenase activity, prostaglandin and leukotriene synthesis, and the activation of the transcription factor NF-kappaB. RESULTS: SK&F 106615 and SK&F 106610 significantly reduced inflammation in the AA- and PMA-induced inflammation models (p < 0.05) with ED50's of 179 and 120 mg/ear for edema and myeloperoxidase, respectively. The compounds did not inhibit eicosanoid biosynthesis, have a direct effect on 5-lipoxygenase or cyclooxygenase enzymes, or inhibit NF-kappaB. CONCLUSIONS: The potent anti-inflammatory and immunomodulatory activities of the azaspiranes observed in these and other studies appear to be mediated by a novel mechanism.

Administration, Topical↗

PubMed1998

Light gradients in spherical photosynthetic vesicles.

Light-gradient photovoltage measurements were performed on EDTA-treated thylakoids and on osmotically swollen thylakoids (blebs), both of spherical symmetry but of different sizes. In the case of EDTA vesicles, a negative polarity (due to the normal light gradient) was observed in the blue range of the absorption spectrum, and a positive polarity, corresponding to an inverse light gradient, was observed at lambda = 530 and lambda = 682 nm. The sign of the photovoltage polarity measured in large blebs (swollen thylakoids) is the same as that obtained for whole chloroplasts, although differences in the amplitudes are observed. An approach based on the use of polar coordinates was adapted for a theoretical description of these membrane systems of spherical symmetry. The light intensity distribution and the photovoltage in such systems were calculated. Fits to the photovoltage amplitudes, measured as a function of light wavelength, made it possible to derive the values of the dielectric constant of the protein, epsilons = 3, and the refractive index of the photosynthetic membrane for light propagating perpendicular and parallel to the membrane surface, nt = 1.42 and nn = 1.60, respectively. The latter two values determine the birefringence of the biological membrane, Deltan = nn - nt = 0.18.

Biophysical Phenomena↗

PubMed1997

Fourier transform infrared difference spectroscopy of photosystem II tyrosine D using site-directed mutagenesis and specific isotope labeling.

Tyrosine D (TyrD), a side path electron carrier of photosystem II (PS II), has been studied by light-induced Fourier transform infrared (FTIR) difference spectroscopy in PS II core complexes of Synechocystis sp. PCC 6803 using the experimental conditions previously optimized to generate the pure TyrD./TyrD FTIR difference spectrum in PS II-enriched membranes of spinach [Hienerwadel, R., Boussac, A., Breton, J., and Berthomieu, C. (1996) Biochemistry 35, 115447-115460]. IR modes of TyrD and TyrD. have been identified by specific 2H- or 13C-labeling of the tyrosine side chains. The v8a(CC) and v19(CC) IR modes of TyrD are identified at 1615 and 1513-1510 cm-1, respectively. These frequencies show that TyrD is protonated. Comparison of isotope-sensitive signals in situ with those of the model compound p-methylphenol dissolved in different solvents leads to the assignment of the v7'a(CO) and delta(COH) modes of TyrD at 1275 and 1250 cm-1, respectively. It is shown that these modes and in particular the delta(COH) IR mode are very sensitive to the formation of hydrogen-bonded complexes with amide C=O or with imidazole nitrogen atoms. The frequencies observed in situ show that TyrD is hydrogen-bonded to the imidazole ring of a neutral histidine. For the radical TyrD., isotope-sensitive IR modes are identified at 1532 and 1503 cm-1. The signal at 1503 cm-1 is assigned to the v(CO) mode of TyrD. since it is sensitive to 13C-labeling at the ring carbon involved in the C4-O bond. The perturbation of TyrD and TyrD. IR modes upon site-directed replacement of D2-His189 by Gln confirms that a hydrogen bond exists between both TyrD and TyrD. and D2-His189. In the D2-His189Gln mutant, the v7'a(CO) mode of TyrD at 1267 cm-1 and the delta(COH) mode at approximately 1228 cm-1 show that a hydrogen bond is formed between TyrD and an amide carbonyl, probably that of the D2-Gln189 side chain. Electron nuclear double resonance (ENDOR) measurements have shown that TyrD. is hydrogen-bonded in the wild type but not in the mutant [Tang, X.-S., Chrisholm, D. A., Dismukes, G. C., Brudwig, G. W., and Diner, B. A. (1993) Biochemistry 32, 13742-13748]. The v(CO) mode of TyrD. at 1497 cm-1 is downshifted by 6 cm-1 compared to WT PS II, indicating that hydrogen bonding induces a frequency upshift of the v(CO) IR mode of Tyr.. IR signals from the Gln side chain v(C=O) mode are proposed to contribute at 1659 and 1692 cm-1 in the TyrD and TyrD. states, respectively. These frequencies are consistent with the rupture of a hydrogen bond upon TyrD. formation in the mutant. The frequency of the v(CO) mode of TyrD., observed at 1503 cm-1 for WT PS II, is intermediate between that observed at 1497 cm-1 in the D2-His189Gln mutant and at 1513 cm-1 for Tyr. formed by UV irradiation in borate buffer, suggesting weaker or fewer hydrogen bonds for TyrD. in PS II than in solution. The role of D2-His189 in proton uptake upon TyrD. formation is also investigated.

Bacterial Proteins↗

PubMed1997

Efficient exchange of the primary quinone acceptor Q(A) in isolated reaction centers of Rhodopseudomonas viridis.

A key step in the conversion of solar energy into chemical energy by photosynthetic reaction centers (RCs) occurs at the level of the two quinones, Q(A) and Q(B), where electron transfer couples to proton transfer. A great deal of our understanding of the mechanisms of these coupled reactions relies on the seminal work of Okamura et al. [Okamura, M. Y., Isaacson, R. A., & Feher, G. (1975) Proc. Natl. Acad. Sci. USA 88, 3491-3495], who were able to extract with detergents the firmly bound ubiquinone Q(A) from the RC of Rhodobacter sphaeroides and reconstitute the site with extraneous quinones. Up to now a comparable protocol was lacking for the RC of Rhodopseudomonas viridis despite the fact that its Q(A) site, which contains 2-methyl-3-nonaprenyl-1,4-naphthoquinone (menaquinone-9), has provided the best x-ray structure available. Fourier transform infrared difference spectroscopy, together with the use of isotopically labeled quinones, can probe the interaction of Q(A) with the RC protein. We establish that a simple incubation procedure of isolated RCs of Rp. viridis with an excess of extraneous quinone allows the menaquinone-9 in the Q(A) site to be almost quantitatively replaced either by vitamin K(1), a close analogue of menaquinone-9, or by ubiquinone. To our knowledge, this is the first report of quinone exchange in bacterial photosynthesis. The Fourier transform infrared data on the quinone and semiquinone vibrations show a close similarity in the bonding interactions of vitamin K(1) with the protein at the Q(A) site of Rp. viridis and Rb. sphaeroides, whereas for ubiquinone these interactions are significantly different. The results are interpreted in terms of slightly inequivalent quinone-protein interactions by comparison with the crystallographic data available for the Q(A) site of the two RCs.

Journal Article↗

PubMed1997

Spectral equilibration and primary photochemistry in Heliobacillus mobilis at cryogenic temperature.

We performed multicolor femtosecond transient absorption measurements on membranes of the photosynthetic bacterium Heliobacillus mobilis at 20 K, by selective excitation at either the red or the blue extreme of the bacteriochlorophyll g Q(Y) band, which is split in three spectral forms (Bchl g 778, 793, and 808) at low temperature. In contrast to room temperature, there is no observable uphill energy transfer upon excitation at the red extreme. This provides a direct experimental confirmation of the expected strong temperature dependence of uphill energy transfer in multichromophore systems. Upon excitation at the blue edge, downhill energy transfer is observed on time ranges varying over 2 orders of magnitude and is discussed in terms of four distinct energy transfer processes: Bchl g 778* --> Bchl g 793* (approximately 50 fs); Bchl g 778* --> Bchl g 808* (approximately 400 fs); Bchl g 793* --> Bchl g 808* (approximately 1.4 ps); and within Bchl g 808* (approximately 7 ps). Surprisingly, the amount of oxidized primary donor P798+ formed on the time scale of picoseconds and tens of picoseconds was found to depend on the excitation conditions: trapping occurs mainly in approximately 80 ps and slower from directly excited Bchl g 808* and can additionally occur in a few picoseconds from Bchl g 778* and Bchl g 793* upon blue excitation. This finding implies that spectral equilibration is not complete prior to charge separation and furthermore is inconsistent with a funnel model, in which P798 is surrounded by long-wavelength pigments. More generally, we discuss to what extent our data bring constraints on the spatial distribution of the different spectral forms of the pigments.

Bacteria↗

PubMed1997

Cloning and recombinant expression of rat and human kynureninase.

Kynureninase [E.C.3.7.1.3.] is one of the enzymes involved in the biosynthesis of NAD cofactors from tryptophan through the kynurenine pathway. By tryptic and CNBr digestion of purified rat liver kynureninase, we obtained about 28% of the amino acid sequence of the enzyme. The rat kynureninase cDNA, isolated by means of reverse-transcribed polymerase chain reaction and hybridization screening, codes for a polypeptide of 464 amino acids. Northern blot analysis revealed the synthesis of a 2.0 kb rat kynureninase mRNA. A cDNA encoding human liver kynureninase was also isolated. The deduced amino acid sequence is 85% identical to that of the rat protein. COS-1 cells were transfected with both cDNAs. The Km values of the rat enzyme, for L-kynurenine and DL-3-hydroxykynurenine, were 440 +/- 20 microM and 32 +/- 5 microM and of the human enzyme 440 /- 20 microM and 49 +/- 6 microM, respectively. Interestingly, COS-1 cells transfected with the cDNA coding for rat kynureninase also display cysteine-conjugate beta-lyase activity.

Amino Acid Sequence↗