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Biomedical subjects

A Boussac

Publications and source records attributed to A Boussac.

34 records · Page 2Linked to original sources
PubMed1995

ESEEM study of the plastoquinone anion radical (QA.--) in 14N- and 15N-labeled photosystem II treated with CN.

The nonheme iron of the photosystem II reaction center was converted to its low-spin state (S = 0) by treatment with CN-. This allowed the study of the plastoquinone, QA- anion radical by electron spin-echo envelope modulation (ESEEM) spectroscopy. A comparative analysis of the ESEEM data of QA- in 14N- and 15N-labeled PSII demonstrates the existence of a protein nitrogen nucleus coupled to the QA-. The 14N coupling is characterized by a quadrupolar coupling constant e2qQ/4h = 0.82 MHz, an asymmetry parameter eta = 0.45, and hyperfine coupling constant A approximately 2.1 MHz. The 15N hyperfine coupling is characterized by T = 0.41 MHz and alpha iso approximately 3.3 MHz. The possible origins of the nitrogen hyperfine coupling are discussed in terms of the amino acids thought to be close to the QA- in PSII. Based on a comparison of the 14N ESEEM with 14N-NQR and 14N-ESEEM data from the literature, the most likely candidate is the amide nitrogen of the peptide backbone of Ala261 of the polypeptide D2, although the indole nitrogen of Trp254 and the imino nitrogen of His215 of D2 also remain candidates.

Cyanides↗
PubMed1995

Histidine oxidation in the S2 to S3 transition probed by FTIR difference spectroscopy in the Ca(2+)-depleted photosystem II: comparison with histidine radicals generated by UV irradiation.

FTIR difference and EPR spectroscopies were used to identify the organic radical species formed during the S2 to S3 transition in Ca(2+)-depleted, EGTA-treated, and polypeptide-reconstituted photosystem II membranes (denoted S2' and S3', respectively). Ferricyanide was added to the samples to act as an exogenous electron acceptor. Using EPR spectroscopy, it was shown that, under the experimental conditions used, only the species oxidized in the S3' state was detected during the time required for the acquisition of the FTIR difference spectra. No contributions from the electron acceptor side were observed. The corresponding S3'/S2' FTIR difference spectra were recorded at 10 degrees C in H2O, D2O, and with 15N-labeled photosystem II membranes. Spectra were compared with radical-minus-neutral FTIR difference spectra of amino acid model compounds generated by UV irradiation at low temperature. Under our experimental conditions, we did not observe FTIR difference signals consistent with tyrosine oxidation in the S2' to S3' transition. The infrared signals characteristic of radical formation with 4-methylimidazole and histidine obtained by UV irradiation of 4-methylimidazolium at pH 6 and of a His-Tyr dipeptide at pH 7 are presented. The analogy found between these spectra and the S3'/S2' spectrum obtained in situ supports the oxidation of a histidinium in the S2' to S3' transition.

Calcium↗
PubMed1994

Direct vibrational structure of protein metal-binding sites from near-infrared Yb3+ vibronic side band spectroscopy.

Near-infrared Yb3+ vibronic side band (VSB) spectroscopy is used to obtain structural information of metal binding sites in metalloproteins. This technique provides a selective "IR-like" vibrational spectrum of those ligands chelated to the Yb3+ ion. VSB spectra of various model complexes of Yb3+ representing different ligand types were studied to provide references for the VSB spectra of Yb(3+)-reconstituted metalloproteins. Ca2+ in the calcium-binding protein parvalbumin and Fe3+ in the iron-transporting protein transferrin were replaced with Yb3+. The fluorescence of Yb3+ reconstituted into these two proteins exhibits weak VSBs whose energy shifts, with respect to the main 2F5/2-->2F7/2 Yb3+ electronic transition, represent the vibrational frequencies of the Yb3+ ligands. The chemical nature of the ligands of the Yb3+ in these proteins, as deduced by the observed VSB frequencies, is entirely in agreement with their known crystal structures. For transferrin, replacement of the 12CO3(2-) metal counterion with 13CO3(2-) yielded the expected isotopic shift for the VSBs corresponding to the carbonate vibrational modes. This technique demonstrates enormous potential in elucidating the localized structure of metal binding sites in proteins.

Apoproteins↗
PubMed1994

Chloride-depletion effects in the calcium-deficient oxygen-evolving complex of photosystem II.

The effects of Cl-depletion in photosystem II (PS-II)-enriched membranes have been investigated by electron paramagnetic resonance (EPR) spectroscopy after removal of the 17- and 23-kDa polypeptides and depletion of Ca2+ by NaCl treatment. When the salt treatment was done in the presence of a high concentration (5 mM) of the chelator [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), a modified dark-stable multiline signal was observed from the S2 state and a 13 mT wide S3 signal could be generated by illumination at 0 degrees C as reported previously for experiments conducted under these conditions [Boussac, A., Zimmermann, J.-L., & Rutherford, A. W. (1990) FEBS Lett. 277, 69-74]. The modified S2 multiline signal was lost after a further Cl- depletion in the presence of a low EGTA concentration (50 microM). Upon Cl- reconstitution, a normal S2 multiline signal could be generated by continuous illumination at 200 K. In contrast, a lowering of the EGTA concentration (50 microM) alone, in the presence of Cl- (30 mM), had no effect on the modified S2 multiline signal. These results indicate that the modification of S2 is due to binding of the chelator to PS-II and that Cl- stabilizes the chelator binding. When Cl- depletion in Ca(2+)-depleted PS-II was done in the presence of a high concentration of EGTA (5 mM), the modified S2 multiline signal disappeared but was regenerated by Cl- reconstitution in darkness. These results indicate that when Cl- depletion is done to the EGTA-modified PS-II, the S2 multiline signal disappears but the S2 state remains stable in the dark.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkanesulfonic Acids↗
PubMed1994

Electron transfer events in chloride-depleted photosystem II.

Cl- and Ca2+ are obligatory cofactors in photosystem II (PS-II), the oxygen-evolving enzyme of plants. The electron transfer events in Cl(-)-depleted PS-II were investigated by using continuous wave (cw) and pulsed EPR, and the results were compared with those obtained in untreated and Ca(2+)-depleted PS-II. In Cl(-)-depleted PS-II, the S1 to S2 transition is not accompanied by the appearance of the S2 manganese signal in both cw and field-swept echo experiments. In the S3 state, the cw experiment reveals the presence of a radical signal, and the field-swept echo experiment reveals the presence of a manganese signal similar to that observed in the S2 state in Cl(-)-reconstituted PS-II. A relaxation enhancement study of the oxidized tyrosine D indicates that a relaxation enhancement occurs only in the S2 to S3 transition and not in the S1 to S2 transition. The results are interpreted by the following preferred model. In Cl(-)-depleted PS-II, the S1 to S2 transition corresponds to the oxidation of a component other than the manganese cluster, possibly an amino acid, and the S2 to S3 transition corresponds to the oxidation of the manganese complex. This oxidation sequence is the opposite of that observed in Ca(2+)-depleted PS-II and may be due to a modulation by chloride of the relative redox potentials of the manganese cluster and a nearby oxidizable amino acid. An alternative model involving manganese oxidation that is invisible in the S1 to S2 transition but that becomes visible on S3 formation cannot be ruled out, although it is considered less likely.

Amino Acids↗
PubMed1993

The manganese center of oxygen-evolving and Ca(2+)-depleted photosystem II: a pulsed EPR spectroscopy study.

The environment of the multi-manganese center in the O2-evolving complex (OEC) of plant photosystem II (PS II) under conditions of Ca2+ depletion has been probed using pulsed electron paramagnetic resonance (EPR) spectroscopy, and the following results are reported: (1) In Ca(2+)-depleted PS II membranes treated with the chelator [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA), the modified Mn EPR signal arising from the OEC in the S2 state and the split EPR signal from the S3 state could be detected in the absorption mode by recording the amplitude of a two-pulse echo as a function of the external magnetic field. The formation of the S3 signal (g approximately 2.004; delta Hpp = 164 G) is not accompanied by the disappearance of the Mn EPR signal, although the signal becomes difficult to detect in CW EPR. This result supports the previous interpretation of the split S3 EPR signal as arising from the interaction of an organic radical with the Mn cluster [Boussac, A., Zimmermann, J. L., Rutherford, A. W., & Lavergne, J. (1990) Nature 347, 303-306]. (2) The two-pulse electron spin echo envelope modulation (ESEEM) spectra of the S2 state formed in Ca(2+)-depleted PS II membranes obtained from 14N- and 15N-labeled material are different. This indicates that nitrogen nuclei from nitrogen-containing protein residues are coupled to the Mn center in the S2 state of the inhibited enzyme. In addition, comparison with the two-pulse ESEEM data obtained for the S2 state in the untreated enzyme suggests that the coupling may be altered by the Ca2+ depletion and/or EGTA treatment. (3) The treatment of Ca(2+)-depleted PS II membranes with sodium pyrophosphate also induced a stable S2 state characterized by a modified multiline EPR signal that is similar to that obtained in EGTA-treated PS II membranes. Comparison of the ESEEM data obtained for the pyrophosphate and 14N and 15N samples treated with EGTA suggests that the modification induced by the EGTA treatment is accompanied by the binding of (an) EGTA molecule(s) to or near the Mn center. (4) ESEEM data obtained for the S3 state formed in the pyrophosphate or EGTA-treated enzyme are quite similar to those obtained for the corresponding S2 state. The data are also compared with ESEEM data obtained on oxidized 4(5)-methylimidazole obtained by UV irradiation. These results are discussed with respect to the current assignment of the S3 radical as arising from oxidation of a histidine residue.

Calcium↗
PubMed1992

Molecular changes following oxidoreduction of cytochrome b559 characterized by Fourier transform infrared difference spectroscopy and electron paramagnetic resonance: photooxidation in photosystem II and electrochemistry of isolated cytochrome b559 and iron protoporphyrin IX-bisimidazole model compounds.

The vibrational infrared absorption changes associated with the oxidation of cytochrome b559 (Cyt b559) have been characterized. In photosystem II (PS II) enriched membranes, low-potential (LP) and high-potential (HP) Cyt b559 were investigated by light-induced FTIR difference spectroscopy. The redox transition of isolated Cyt b559 is characterized by protein electrochemistry. On the basis of a model of the assembly of Cyt b559 with the two axial Fe ligands being histidine residues of two distinct polypeptides, each forming a transmembrane alpha-helix [Cramer, W.A., Theg, S.M., & Widger, W.R. (1986) Photosynth. Res. 10, 393-403], the bisimidazole and bismethylimidazole complexes of Fe protoporphyrin IX were electrochemically oxidized and reduced to detect the IR oxidation markers of the heme and its two axial ligands. Major bands at 1674/1553, 1535, and 1240 cm-1 are tentatively assigned to nu 37 (CaCm), nu 38-(CbCb) and delta (CmH) modes, respectively; other bands at 1626, 1613, 1455, 1415, and 1337 cm-1 are assigned to porphyrin skeletal and vinyl modes. Modes at 1103 and 1075/1066 cm-1 are assigned to the 4-methylimidazole and imidazole ligands, respectively. For the isolated Cyt b559, it is shown that both the heme (at 1556-1535, 1337, and 1239 cm-1), the histidine ligands at 1104 cm-1 and the protein (between 1600 and 1700 cm-1 and at 1545 cm-1) are affected by the charge stabilization. The excellent agreement between model compounds and isolated Cyt b559 reinforces the validity of the model of a heme iron coordinated to two histidine residues for Cyt b559. A differential signal at 1656/1641 cm-1 is assigned to peptide C = O mode(s). We speculate that this signal reflects the change in strength of a hydrogen bond formed between the histidine ligand(s) and the polypeptide backbone upon oxidoreduction of the cytochrome. In PS II membranes, the signals characteristic of Cyt b559 photooxidation are found at 1660/1652 and 1625 cm-1, for both the high- and low-potential forms. The differences observed in the amplitude of the 1660/1652-cm-1 band, at 1700 and 1530-1510 cm-1 in the light-induced FTIR difference spectra of Cyt b559 HP and LP, show that the mechanisms of heme oxidation in vivo imply different molecular processes for the two forms Cyt b559 HP and LP.

Cold Temperature↗
PubMed1992

The origin of the split S3 EPR signal in Ca(2+)-depleted photosystem II: histidine versus tyrosine.

The radical formed as the formal S3 charge storage state in Ca(2+)-depleted photosystem II and detected as a split EPR signal was previously assigned to an oxidized histidine radical on the basis of its UV spectrum. In a recent paper [Hallahan, B. J., Nugent, J. H. A., Warden, J. T., & Evans, M. C. W. (1992) Biochemistry 31, 4562-4573], this assignment was challenged, and it was suggested that the signal arises instead from the well-known tyrosine radical Tyrz., the electron carrier between the photooxidized chlorophyll and the Mn cluster. Here, we provide evidence that the measurements of the Tyr., on which the new interpretation was based, are artifactual due to the use of saturating microwave powers. Other than a relaxation-enhancement effect, the formation of the split S3 signal is accompanied by no change in the Tyr. signal. Although essentially unrelated to the origin of the S3 radical, several other experimental and interpretational problems in the work of Hallahan et al. (1992) are pointed out and rationalized. For example, the inability of Hallahan et al. (1992) to observe the split S3 signal in samples containing DCMU or without a chelator, in contrast to our observations, is attributed to a number of technical problems including the incomplete inhibition of the enzyme. We thus conclude that the assignment of the split S3 signal as His., although not proven, remains the most reasonable on the basis of current data.

Calcium↗
PubMed1992

Near-infrared Yb(3+) vibronic sideband spectroscopy: application to Ca(2+)-binding proteins.

We have used near-infrared (NIR) vibronic fluorescence spectroscopy to study the vibrational structure of ligands associated with model complexes of the lanthanide Yb(3+). This technique exploits the similar binding properties of the lanthanide Yb(3+) to probe Ca(2+)-binding sites in proteins. The (NIR) fluorescence of complexed Yb(3+) exhibits, in addition to main 0-0 (2F5/2----2F7/2) electronic transition of Yb(3+), weak vibronic sidebands which provide infrared-like, local vibrational spectra of the chelates (inner sphere ligands) of Yb(3+). A similar approach has been used for the lanthanide Gd(3+) (MacGregor, R.B., Jr (1989) Arch. Biochem. Biophys. 274, 312-316) which fluoresces in the UV and which is usually complicated by amino-acid residues fluorescing in the same spectral region. In this same spectral region, other complications in studying photosynthetic membranes occur in the form of the excitation wavelength being actinic, promoting photodegradation of the membranes, as well as the reabsorption of Gd(3+) fluorescence. NIR excitation and fluorescence detection of Yb(3+) avoid these problems when studying photosynthetic membranes. A preliminary study has been conducted here on rat muscle parvalbumin.

Animals↗
PubMed1992

Inhibition of tyrosine Z photooxidation after formation of the S3 state in Ca(2+)-depleted and Cl(-)-depleted photosystem II.

Ca2+ and Cl- are obligatory cofactors in photosystem II (PS-II), the oxygen-evolving enzyme of plants. The sites of inhibition in both Ca(2+)- and Cl(-)-depleted PS-II were compared using EPR and flash absorption spectroscopies to follow the extent of the photooxidation of the redox-active tyrosine (TyrZ) and of the primary electron donor chlorophyll (P680) and their subsequent reduction in the dark. The inhibition occurred after formation of the S3 state in Ca(2+)-depleted PS-II. In Cl(-)-depleted photosystem II, the inhibition occurred after formation of the S3 state in about half of the centers and probably after S2TyrZ+ formation in the remaining centers. After the S3 state was formed in Ca(2+)- and Cl(-)-depleted photosystem II, electron transfer from TyrZ to P680 was inhibited. This inhibition is discussed in terms of electrostatic constraints resulting from S3 formation in the absence of Ca2+ and Cl-.

Buffers↗
PubMed1990

Factors influencing the formation of modified S2 EPR signal and the S3 EPR signal in Ca(2+)-depleted photosystem II.

NaCl/EGTA-washing of photosystem II (PS-II) results in the removal of Ca2+ and the inhibition of oxygen evolution. Two new EPR signals were observed in such samples: a stable and modified S2 multiline signal and an S3 signal [(1989) Biochemistry 28, 8984-8989]. Here, we report what factors are responsible for the modifications of the S2 signal and the observation of the S3 signal. The following results were obtained. (i) The stable, modified, S2 multiline signal can be induced by the addition of high concentrations of EGTA or citrate to PS-II membranes which are already inhibited by Ca(2+)-depletion. (ii) The carboxylic acids act in the S3-state, are much less effective in S2 and have no effect in the S1-state. (iii) The extrinsic polypeptides (17- and 23-kDa) are not required to observe either the modified S2 signal or the S3 signal. However, they do influence the splitting and the lifetime of the S3 signal, and they seem to have a slight influence on the hyperfine pattern of the S2 signal. (iv) The S3 signal can be observed in Ca(2+)-depleted PS-II which does not exhibit the modified multiline signal. Then, it is proposed that formation of histidine radical during the S2 to S3 transition in Ca(2+)-depleted PS-II [(1990) Nature 347, 303-306] also occurs in functional PS-II.

Calcium↗
PubMed1990

Interaction of ammonia with the water splitting enzyme of photosystem II.

The effects of NH3 on the oxygen evolving enzyme have been investigated with EPR and steady-state O2 evolution. The following results were obtained. At low light intensity O2 evolution occurs in all centers even though ammonia is bound. This binding occurs in the S2 state and results in a modification of the multiline signal as reported earlier. However, the oscillations with flash number of the amplitude of the EPR signal are virtually unaffected, indicating that NH3 binding does not prevent S-state advancement. Inhibition of O2 evolution by NH3 measured at light intensities that are nearly saturating for untreated photosystem II is interpreted as being due to a slow down in the rate of S-state cycling. At very high light intensities NH3 is not able to inhibit oxygen evolution presumably because NH3 binding is S state dependent and the susceptible S state (S2) is turned over too quickly. NH3 binding resulting in the modified multiline signal does not occur in S1. When S1 is formed from fully NH3 modified S2 by deactivation or by three further flashes, the S1 state does not have NH3 bound. NH3 thus dissociates easily from S1. Earlier reports of NH3 binding in S1 may be explained by the observation that NH3 binding can occur upon incubation of samples in S2 at temperatures as low as 198 K. Evidence is obtained for an NH3 binding occurring slowly (30 s) in S3. This binding results in a block in S-state advancement as suggested earlier [Velthuys, B. R. (1975) Thesis, University of Leiden]. The results are interpreted in two possible models: (1) NH3 binding in S2 occurs in a substrate site, but it is rapidly exchanged by water upon S4 formation. (2) NH3 binding in S2 is not in a substrate site but instead in a structural site and remains bound while water is oxidized. Inherent in this model is that other NH3 binding sites, i.e., the Cl- site, and the slow NH3 binding site in S3 could be the true substrate sites. Some mechanistic implications are discussed.

Ammonia↗
PubMed1989

EPR signals from modified charge accumulation states of the oxygen evolving enzyme in Ca2+-deficient photosystem II.

Photosystem II enriched membranes were depleted of Ca2+ and the 17- and 23-kDa polypeptides by treatment with NaCl and EGTA. The 17- and 23-kDa polypeptides were then reconstituted. This preparation was incapable of O2 evolution until Ca2+ was added. An EPR study revealed the presence of two new EPR signals. One of these is a modified S2 multiline signal with an isotropic g value of 1.96 with at least 26 hyperfine peaks (average spacing 55 G) distributed over approximately 1600 G. The other is a near-Gaussian signal with an isotropic g value of 2.004, which is attributed to a formal S3 state. Experiments involving the interconversion of these signals and the effect of Ca2+ and Sr2+ rebinding provide evidence for these assignments. From these results the following conclusions are drawn: (1) These results are consistent with our earlier demonstration that charge accumulation is blocked after formation of S3 when Ca2+ is deficient. (2) Binding of the 17- and 23-kDa polypeptides to photosystem II in the absence of Ca2+ results in the perturbation of the Mn cluster. This is taken as a further indication that the Ca2+-binding site is close to or even an integral part of the Mn cluster. (3) The S3 signal may arise from an organic free radical interacting magnetically with the Mn cluster. However, other possible origins for this signal, including the Mn cluster itself, must also be considered.

Calcium↗
PubMed1989

Mutagenesis by random cloning of an Escherichia coli kanamycin resistance gene into the genome of the cyanobacterium Synechocystis PCC 6803: selection of mutants defective in photosynthesis.

Photosynthetic mutants of the cyanobacterium Synechocystis PCC 6803 were produced by a random cartridge mutagenesis method leading to gene inactivation. This procedure relies on random ligation of an Escherichia coli kanamycin resistance (Kmr) gene to restriction fragments of genomic DNA from the host. Then recombination occurring during transformation promotes integration of the marker gene into the genome of the recipient cells. Several mutants impaired in photosynthesis were obtained by this procedure. All are partially or totally defective in photosystem II activity and some of them also harbour a functionally modified photosystem I. Restriction and recombination data showed that one mutant (AK1) is best explained as an insertion of the Kmr gene into an AvaII restriction site of the gene psbD-1. All others harbour a deletion, ranging from at least 1.15 kb (AK3) to more than 50 kb (AK9), which partly or fully overlaps the genes psbB and/or psbD-1, depending on the mutant. A genetic-physical map of the more than 60 kb region of the cyanobacterial genome harbouring the genes psbB, psbC and psbD-1 was constructed by combining published sequence data on these genes with the results of recombination and restriction mapping.

Chromosome Mapping↗