Delineation of cellular stages and identification of key proteins for reduction and biotransformation of Se(IV) by Stenotrophomonas bentonitica BII-R7.

The widespread use of selenium (Se) in technological applications (e.g., solar cells and electronic devices) has led to an accumulation of this metalloid in the environment to toxic levels. The newly described bacterial strain Stenotrophomonas bentonitica BII-R7 has been demonstrated to reduce mobile Se(IV) to Se(0)-nanoparticles (Se(0)NPs) and volatile species. Amorphous Se-nanospheres are reported to aggregate to form crystalline nanostructures and trigonal selenium. We investigated the molecular mechanisms underlying the biotransformation of Se(IV) to less toxic forms using differential shotgun proteomics analysis of S. bentonitica BII-R7 grown with or without sodium selenite for three different time-points. Results showed an increase in the abundance of several proteins involved in Se(IV) reduction and stabilization of Se(0)NPs, such as glutathione reductase, in bacteria grown with Se(IV), in addition to many proteins with transport functions, including RND (resistance-nodulation-division) systems, possibly facilitating Se uptake. Notably proteins involved in oxidative stress defense (e.g., catalase/peroxidase HPI) were also induced by Se exposure. Electron microscopy analyses confirmed the biotransformation of amorphous nanospheres to trigonal Se. Overall, our results highlight the potential of S. bentonitica in reducing the bioavailability of Se, which provides a basis both for the development of bioremediation strategies and the eco-friendly synthesis of biotechnological nanomaterials.

Mechanism and dynamics of fatty acid photodecarboxylase.

Fatty acid photodecarboxylase (FAP) is a photoenzyme with potential green chemistry applications. By combining static, time-resolved, and cryotrapping spectroscopy and crystallography as well as computation, we characterized Chlorella variabilis FAP reaction intermediates on time scales from subpicoseconds to milliseconds. High-resolution crystal structures from synchrotron and free electron laser x-ray sources highlighted an unusual bent shape of the oxidized flavin chromophore. We demonstrate that decarboxylation occurs directly upon reduction of the excited flavin by the fatty acid substrate. Along with flavin reoxidation by the alkyl radical intermediate, a major fraction of the cleaved carbon dioxide unexpectedly transformed in 100 nanoseconds, most likely into bicarbonate. This reaction is orders of magnitude faster than in solution. Two strictly conserved residues, R451 and C432, are essential for substrate stabilization and functional charge transfer.

Iron coordination in photosystem II: interaction between bicarbonate and the QB pocket studied by Fourier transform infrared spectroscopy.

The non heme iron environment of photosystem II is studied by light-induced infrared spectroscopy. A conclusion of previous work [Hienerwadel, R., and Berthomieu, C. (1995) Biochemistry 34, 16288-16297] is that bicarbonate is a bidendate ligand of the reduced iron and a monodentate ligand in the Fe(3+) state. In this work, the effects of bicarbonate replacement with lactate, glycolate, and glyoxylate, and of o-phenanthroline binding are investigated to determine the specific interactions of bicarbonate with the protein. Fe(2+)/Fe(3+) FTIR spectra recorded with (12)C- and (13)C(1)-labeled lactate indicate that lactate displaces bicarbonate by direct binding to the iron through one carboxylate oxygen and the hydroxyl group in both the Fe(2+) and Fe(3+) states. This different binding mode with respect to bicarbonate could explain the lower midpoint of the iron couple observed in the presence of this anion [Deligiannakis, Y., Petrouleas, V., and Diner, B. A. (1994) Biochim. Biophys. Acta 1188, 260-270]. In agreement with the -60 mV/pH unit dependence of the iron midpoint potential in the presence of bicarbonate, the proton release upon iron oxidation by photosystem II is directly measured to 0.95 +/- 0.05 by the comparison of infrared signals of phosphate buffer and ferrocyanide modes. This accurate method may be applied to the study of other redox reactions in proteins. The pH dependence of the iron couple is proposed to reflect the deprotonation of D1His215, a putative iron ligand located at the Q(B) pocket, since the signal at 1094 cm(-1) assigned to the nu(C-N) mode of a histidinate ligand in the Fe(3+) state is not observed in the presence of o-phenanthroline. Specific regulation of the pK(a) of D1His215 by bicarbonate is inferred from the absence of the band at 1094 cm(-1) in Fe(2+)/Fe(3+) spectra recorded with glycolate, glyoxylate, or lactate. A broad positive continuum, maximum at approximately 2550 cm(-1), observed in the presence of bicarbonate, but absent with o-phenanthroline or lactate, glycolate, and glyoxylate, indicates a hydrogen bond network from the non heme iron toward the Q(B) pocket involving bicarbonate and His D1-215. Proton release of about 1, measured upon iron oxidation at pH 6 with the latter anions, points to a proton release mechanism different from that involved in the presence of bicarbonate.

NMR conformational study of the sixth transmembrane segment of sarcoplasmic reticulum Ca2+-ATPase.

In current topological models, the sarcoplasmic reticulum Ca2+-ATPase contains 10 putative transmembrane spans (M1-M10), with spans M4/M5/M6 and probably M8 participating in the formation of the membranous calcium-binding sites. We describe here the conformational properties of a synthetic peptide fragment (E785-N810) encompassing the sixth transmembrane span (M6) of Ca2+-ATPase. Peptide M6 includes three residues (N796, T799, and D800) out of the six membranous residues critically involved in the ATPase calcium-binding sites. 2D-NMR experiments were performed on the M6 peptide selectively labeled with 15N and solubilized in dodecylphosphocholine micelles to mimic a membrane-like environment. Under these conditions, M6 adopts a helical structure in its N-terminal part, between residues I788 and T799, while its C-terminal part (G801-N810) remains disordered. Addition of 20% trifluoroethanol stabilizes the alpha-helical N-terminal segment of the peptide, and reveals the propensity of the C-terminal segment (G801-L807) to form also a helix. This second helix is located at the interface or in the aqueous environment outside the micelles, while the N-terminal helix is buried in the hydrophobic core of the micelles. Furthermore, the two helical segments of M6 are linked by a flexible hinge region containing residues T799 and D800. These conformational features may be related to the transient formation of a Schellman motif (L797VTDGL802) encoded in the M6 sequence, which probably acts as a C-cap of the N-terminal helix and induces a bend with respect to the helix axis. We propose a model illustrating two conformations of M6 and its insertion in the membrane. The presence of a flexible region within M6 would greatly facilitate concomitant participation of all three residues (N796, T799, and D800) believed to be involved in calcium complexation.

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.

Effect of (13)C-, (18)O- and (2)H-labeling on the infrared modes of UV-induced phenoxyl radicals.

The structure and environment of redox active tyrosines present in several metalloenzymes can be studied by resonance Raman spectroscopy or Fourier transform infrared difference spectroscopy. Assignments of the vibrational modes in vivo often requires in vitro studies on model compounds. This approach is briefly reviewed. New results are shown on the influence of isotope-labeling on the infrared spectra of tyrosine, [Formula: see text] and phenol radicals obtained in vitro by UV-irradiation. The infrared spectra of the radicals are dominated by the [Formula: see text] mode at 1515-1504 cm(-1). The frequency shifts induced on this mode by (13)C- (2)H-, and (18)O-labeling are reported.

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.

Fourier transform infrared difference study of tyrosineD oxidation and plastoquinone QA reduction in photosystem II.

Two redox active tyrosines are present in the homologous polypeptides D1 and D2 of photo-system II (PS II). TyrZ (D1-161) is involved in the electron transfer reactions resulting in oxygen evolution, while TyrD (D2-160) usually forms a dark-stable radical. In Mn-depleted PS II, TyrD. can be slowly reduced by exogenous reductants. Charge separation then results in the oxidation of TyrD and TyrZ and the reduction of the primary electron acceptor QA. The semiquinone QA- can be reoxidized by oxidants like ferricyanide. In the present work, experimental conditions leading to the generation of pure QA-/QA or TyrD./TyrD FTIR difference spectra have been optimized. Therefore, single-turnover flashes or short illuminations were performed on PS II samples in the presence of exogenous reductants or oxidants. The QA- and TyrD. radicals were generated with high yield and with a lifetime of several seconds or minutes allowing averaging of FTIR difference spectra with high signal to noise ratio. Both QA- formation and contributions at the electron donor side of PS II were monitored by EPR spectroscopy. In PS II samples at pH 6 in the presence of PMS, NH2OH, and DCMU, EPR measurements show that QA- is formed with high yield upon a 1 s illumination at 10 degrees C, while no radical from the electron donor side of PS II is detected. Therefore the QA-/QA FTIR spectrum obtained in these conditions shows only vibrational changes due to QA reduction in PS II. In contrast, a similar spectrum was recently interpreted in terms of dominant contributions from Chl+/Chl signals [MacDonald, G. M., Steenhuis, J. J., & Barry, B. A. (1995) J. Biol. Chem. 270, 8420-8428], although the contribution from the electron acceptor QA was not quantified. In particular, it is shown here that the large positive signal at 1478 cm-1 is due to the QA- state and not to a Chl+ mode. This band is not downshifted upon 15N-labeling of spinach PS II membranes within the +/- 1 cm-1 accuracy of the method and is therefore tentatively assigned to the v(C[symbol: see text]O) mode of the plastosemiquinone QA-. Also unchanged upon 15N-labeling, signals at 1644 and/or 1630 cm-1 are possible candidates for the v(C = O) mode(s) of neutral QA in PS II. The TyrD./TyrD FTIR spectrum is recorded at 4 degrees C on Tris-washed PS II membranes from spinach at pH 6 in the presence of phosphate, formate, and ferricyanide. EPR experiments performed on these samples show that almost all TyrD. is formed upon a 1 s illumination at 4 degrees C and that TyrD. is then reduced within 12 min in the dark. No contributions from TyrZ. or QA- are detected 2 s after illumination. It is thus possible to optimize experimental conditions to record the FTIR difference spectrum only due to TyrD photooxidation in PS II-enriched membranes of spinach. The TyrD./TyrD FTIR spectrum is compared to a cresol./cresol FTIR difference spectrum obtained by UV irradiation at 10 K of cresol at pH 8. The spectral analogies observed between the in vivo and in vitro spectra recorded either in H2O or in D2O suggest that IR modes of TyrD contribute at 1513 and 1252 cm-1. These frequencies are characteristic of a protonated tyrosine. A positive signal is observed at 1506 cm-1 for cresol. and at 1504 cm-1 for the TyrD. state. This suggests contribution of the TyrD. side chain at 1504 cm-1. A band at 1473 cm-1 was previously assigned to the v(CO) mode of TyrD. [MacDonald, G. M., Bixby, K. A., & Barry, B. A. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 11024-11028]. In contrast, no positive signal is observed at 1473 cm-1 in the TyrD./TyrD FTIR difference spectrum presented here. The TyrD./TyrD spectrum also shows vibrational changes from peptide groups and amino acid side chains which are modified upon TyrD. formation. Proton release at the PS II protein surface upon TyrD. formation is deduced from differential signals at the v(PO) modes of phosphate.

Bicarbonate binding to the non-heme iron of photosystem II investigated by Fourier transform infrared difference spectroscopy and 13C-labeled bicarbonate.

The binding site of the non-heme iron of photosystem II (PS II) is investigated by light-induced Fourier tranform infrared (FTIR) difference spectroscopy on Tris-washed membranes. The non-heme iron is oxidized (Fe3+) in the dark with ferricyanide and reduced (Fe2+) after light-induced charge separation by electron transfer from the semiquinone anion QA-. EPR experiments and IR modes of ferri- and ferrocyanide show that the electron donor side of PS II is reduced in less than 2 s after a flash and that ferricyanide reoxidizes the non-heme iron with a half-time of approximately 20 s. Recording FTIR spectra before and 2 s after flash illumination thus results in the Fe2+/Fe3+ difference spectrum. This spectrum shows band shifts and intensity changes of IR modes from ligands and neighboring residues of the non-heme iron. The IR modes of bicarbonate are revealed by comparison of Fe2+/Fe3+ spectra obtained on PS II membranes with 12C or 13C isotope labeled bicarbonate in H2O and in 2H2O. The nu as(CO) and nu s(CO) modes of bicarbonate in the Fe2+ state are assigned at 1530 +/- 10 and 1338 cm-1, respectively. The low frequency of the nu as(CO) mode is taken as experimental evidence that bicarbonate is a ligand of the non-heme iron. Furthermore, the small frequency difference (192 cm-1) between the nu as(CO) and nu s(CO) modes as compared to even hydrogen-bonded ionic bicarbonate strongly indicates that bicarbonate is a bidentate ligand of the non-heme iron in PS II. Upon iron oxidation, the bicarbonate modes are largely affected. The nu s(CO) mode is assigned at 1228 cm-1, while the nu as(CO) mode is tentatively assigned at 1658 +/- 20 cm-1. The strong up- and downshifts of the nu as and nu s(CO) modes of bicarbonate upon iron oxidation results in a frequency difference of 430 +/- 20 cm-1 that is not only explained by the increased charge on the iron but indicates that bicarbonate is a monodentate ligand of the oxidized iron. The sensitivity of the nu s(CO) mode of bicarbonate to 1H/2H exchange in both the Fe2+ and Fe3+ states and the presence in the Fe2+ state of a delta (COH) mode at 1258 cm-1 confirm that bicarbonate and not carbonate is the iron ligand and further exhibits hydrogen bond(s) with the protein. The 13C isotope-sensitive modes of bicarbonate are not affected by 15N labeling of the PS II membranes. 15N sensitive signals at 1111/1102 and 1094 cm-1 are assigned to side chain modes from histidine ligands of the iron. The latter signal is proposed to account for a histidine ligand that deprotonates upon iron oxidation. The involvement of protein peptide groups and side chains in the hydrogen-bond network around the iron is also discussed.

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.

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)

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.

Photooxidation of high-potential (c559, c556) and low-potential (c552) hemes in the cytochrome subunit of Rhodopseudomonas viridis reaction center. Characterization by FTIR spectroscopy.

The photooxidation of c559, c556 and c552 hemes in Rhodopseudomonas viridis cytochrome has been characterized by light-induced FTIR difference spectroscopy. Apart from the common features at 1659 cm-1 and 1561/1551 cm-1 which could arise from one (or possibly two) peptide bond(s), no evidence for major structural rearrangement of the polypeptide backbone was observed. A significant difference with respect to redox-induced FTIR spectra of cytochrome c is the absence of the Tyr marker at 1514/1518 cm-1 in Rps. viridis cytochrome, indicating that the localized shift of a Tyr side chain observed between ferro- and ferri-cytochrome c does not occur in Rps. viridis cytochrome.

Probing the secondary quinone (QB) environment in photosynthetic bacterial reaction centers by light-induced FTIR difference spectroscopy.

The photoreduction of the secondary electron acceptor, QB, has been characterized by light-induced Fourier transform infrared difference spectroscopy of Rb. sphaeroides and Rp. viridis reaction centers. The reaction centers were supplemented with ubiquinone (UQ10 or UQ0). The QB- state was generated either by continuous illumination at very low intensity or by single flash in the presence of redox compounds which rapidly reduce the photooxidized primary electron donor P+. This approach yields spectra free from P and P+ contributions making possible the study of the microenvironment of QB and QB-. Assignments are proposed for the C...O vibration of QB- and tentatively for the C = O and C = C vibrations of QB.

Probing the primary quinone environment in photosynthetic bacterial reaction centers by light-induced FTIR difference spectroscopy.

The photoreduction of the primary electron acceptor, QA, has been characterized by light-induced Fourier transform infrared difference spectroscopy for Rb. sphaeroides reaction centers and for Rsp. rubrum and Rp. viridis chromatophores. The samples were treated both with redox compounds, which rapidly reduce the photooxidized primary electron P+, and with inhibitors of electron transfer from QA- to the secondary quinone QB. This approach yields spectra free from P and P+ contributions which makes possible the study of the microenvironment of QA and QA-.

Characterization by FTIR spectroscopy of the photoreduction of the primary quinone acceptor QA in photosystem II.

Molecular changes associated with the photoreduction of the primary quinone acceptor Qa of photosystem II have been characterized by Fourier transform infrared spectroscopy. This reaction was light-induced at room temperature on photosystem II membranes in the presence of hydroxylamine and diuron. A positive signal at 1478 cm-1 is assigned to the C---O stretching mode of the semiquinone anion, and can be correlated to the negative C=O mode(s) of the neutral QA at 1645 cm-1 and/or 16 cm-1. Analogies with bacterial reaction center are found in the amide I absorption range at 1672 cm-1, 1653 cm-1 and 1630 cm-1. The stabilization of QA- does not result from a large protein conformation change, but involves perturbations of several amino acid vibrations. At 1658 cm-1, a negative feature sensitive to 1H-2H exchange is tentatively assigned to a NH2 histidine mode, while tryptophan D2252 could contribute to the signal at 1560/1550 cm-1.