Eliane Nabedryk

A protein conformational change associated with the photoreduction of the primary and secondary quinones in the bacterial reaction center

A comparison is made between the PQA → P+Q− A and PQAQB → P+QAQ− B transitions in Rps. viridis and Rb. sphaeroides reaction centers (RCs) by the use of light‐induced Fourier transform infrared (FTIR) difference spectroscopy. In Rb. sphaeroides RCs, we identify a signal at 1650 cm−1 which is present in the P+QA‐minus‐PQA spectrum and not in the P+QAQ− B‐minus‐PQAQB spectrum. In contrast, this signal is present in both P+Q− A‐minus‐PQ− A and P+QAQ− B‐minus‐PQAQB spectra of Rps. viridis RCs. These data are interpreted in terms of a conformational change of the protein backbone near QA (possible at the peptide C≡O of a conserved alanine residue in the QA pocket) and of the different bonding interactions of QB with the protein in the RC of the two species.

Light-induced Fourier transform infrared (FTIR) spectroscopic investigations of the primary donor oxidation in bacterial photosynthesis

Fourier transform infrared (FTIR) difference spectroscopy of the primary electron donor (P) photo–oxidation has been performed for reaction centers (RCs) and chromatophores of purple photosynthetic bacteria. In the 1800–650 cm−1 spectral region highly reproducible absorbance changes were obtained that can be related to specific changes of individual bond absorption. Several bands in the difference spectra are tentatively assigned to changes of intensity and position of the keto and ester C−O vibrations of the P bacteriochlorophylls, and a possible interpretation in terms of changes of their environment or type of bonding to the protein is given. Small difference bands in the amide I and II region allow only minor protein conformational changes.

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 δNH2 histidine mode, while tryptophan D2252 could contribute to the signal at cm−1.

CHARACTERIZATION OF BONDING INTERACTIONS OF THE INTERMEDIARY ELECTRON ACCEPTOR IN THE REACTION CENTER OF PHOTOSYSTEM II BY FTIR SPECTROSCOPY

Abstract Molecular changes associated with the photoreduction of the pheophytin a intermediary electron acceptor in films of Photosystem II reaction center (D1D2 RC) were characterized by FTIR spectroscopy. Upon accumulation at 240 K of the photoreduced acceptor, three negative carbonyl bands are observed at 1739 cm−1, 1721 cm−1 and 1677 cm−1 in the light-minus-dark FTIR spectrum of D1D2 RC. The redox-induced FTIR spectrum of the pheophytin a anion generated electrochemically in tetrahydrofuran shows only two negative bands at 1743 cm−1 and 1706 cm−1 which are assigned to changes of absorption of the 10a-ester C=O and 9-keto C=O, respectively. These assignments are based upon the comparison between FTIR data obtained on radicals of pheophytin a and its pyroderivative lacking the 10a-ester C=O. Thus, the 1677 cm−1 band observed in vivo reflects an interacting 9-keto C=O in D1D2 RC. The close similarity observed between: (i) FTIR spectra obtained on Photosystem II and Rps. viridis reaction centers and (ii) amino-acid sequences of the L and D1 polypeptides leads to the assignment of the 1721 cm−1 band in D1D2 RC to a protein-bound 10a-ester C=O of the acceptor and the 1739 cm−1 band to a contribution from the protonated carboxylic group of Glu D1-130 which is proposed to be H-bonded to the 9-keto C=O of the pheophytin acceptor, in the same way as in the Rps. viridis reaction center, Glu L104 is interacting with the 9-keto C=O of HL. The FTIR data indicate that the interactions of the 9-keto C=O and of the 10a-ester of the intermediary acceptor with the protein are stronger in D1D2 RC than in Rps. viridis. These stronger interactions could account, at least in part, for the difference in accessibility to 1H-2H exchange of the H-bonded proton of the Glu D1-130 side-chain in D1D2 RC compared to Rps. viridis reaction center.

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.

Investigation of models for photosynthetic electron acceptors : infrared spectroelectrochemistry of ubiquinone and its anions

In order to gain more information about the binding and interaction of quinones acting as electron acceptors in photosynthetic reaction centers, infrared spectra of ‘model” quinones and their reduced species (semiquinones, quinone dianions and quinols) were recorded in a spectroelectrochemical cell. The frequencies and the extinction coefficients of the quinone C = O and C = C stretching vibrations which are shifted upon reduction were obtained in different solvents. These spectra are used to estimate the contribution of quinone reduction to light‐induced infrared difference spectra between the charge‐separated and the relaxed state of photosynthetic reaction centers.

Light-induced Fourier transform infrared (FTIR) spectroscopic investigations of primary reactions in photosystem I and photosystem II

Light‐induced Fourier transform infrared (FTIR) difference spectroscopy has been applied for the first time to primary reactions in green plant photosynthesis. Photooxidation of the primary electron donor (P700) in photosystem I‐enriched particles as well as in thylakoids, and photoreduction of the pheophytin (Pheo) intermediary electron acceptor in photosystem II‐enriched particles, have led to reproducible difference spectra. In the spectral range investigated (between 1800 and 1000 cm−1) several bands are tentatively attributed to changes in intensity and position of the keto and ester carbonyl vibrations of the chlorophyll or Pheo molecule(s) involved. For some of these groups, possible interpretations in terms of changes of their environment or type of bonding to the protein are given. The intensity of the differential features in the amide I and amide II spectral region allows the exclusion of the eventuality of large protein conformational changes occurring upon primary charge separation.

Transmembrane orientation of α-helices in the thylakoid membrane and in the light-harvesting complex. A polarized infrared spectroscopy study

Abstract The structure and orientation of the major protein constituent of photosynthetic membranes in green plants, the chlorophyll a b light-harvesting complex (LHC) have been investigated by ultraviolet circular dichroism (CD) and polarized infrared spectroscopies. The isolated purified LHC has been reconstituted into phosphatidylcholine vesicles and has been compared to the pea thylakoid membrane. The native orientation of the pigments in the LHC reconstituted in vesicles was characterized by monitoring the low-temperature polarized absorption and fluorescence spectra of reconstituted membranes. Conformational analysis of thylakoid and LHC indicate that a large proportion of the thylakoid protein is in the α-helical structure (56 ± 4%), while the LHC is for 44 ± 7% α-helical. By measuring the infrared dichroism of the amide absorption bands of air-dried oriented multilayers of thylakoids and LHC reconstituted in vesicles, we have estimated the degree of orientation of the α-helical chains with respect to the membrane normal. Infrared dichroism data demonstrate that transmembrane α-helices are present in both thylakoid and LHC with the α-helix axes tilted at less than 30° in LHC and 40° in thylakoid with respect to the membrane normal. In thylakoids, an orientation of the polar C=O ester groups of the lipids parallel to the membrane plane is detected. Our results are consistent with the existence of 3–5 transmembrane α-helical segments in the LHC molecules.

Orientation of intrinsic proteins in photosynthetic membranes. Polarized infrared spectroscopy of chloroplasts and chromatophores

In order to estimate the degree of orientation of the alpha-helices of intrinsic proteins in photosynthetic membranes, polarized infrared spectroscopy has been used to measure the dichroism of the amide I and amide II absorption bands of air-dried oriented samples of purple membranes, chloroplasts and chromatophores from Rhodopseudomonas sphaeroides. Using purple membrane, in which the orientation of the alpha-helices is precisely known (Henderson, R. (1977) Annu. Rev. Biophys. Bioeng. 6, 87-109), as a standard to calibrate our measurements and estimating the mosaic spread (extent of orientation) of the membranes from linear dichroism measurements performed in the visible spectral range, it is concluded that in photosynthetic membranes, the alpha-helices of intrinsic proteins are tilted at less than 40 degrees with respect to the normal to the plane of the membrane.

LIGHT‐INDUCED FOURIER TRANSFORM INFRARED SPECTROSCOPIC INVESTIGATIONS OF THE INTERMEDIARY ELECTRON ACCEPTOR REDUTION IN BACTERIAL PHOTOSYNTHESIS

Abstract— Molecular changes associated with the light‐induced reduction of the intermediary electron acceptor I (bacteriopheophytin, BPh) in bacterial photosynthesis were studied by means of Fourier transform infrared (FTIR) difference spectroscopy. Chromatophore membranes and reconstituted reaction centers (RCs) of Rhodopseudomonas viridis were prereduced with sodium dithionite and illuminated in order to trap photochemically the state I−. Fourier transform infrared spectra of these samples were recorded before, during and after illumination, with an accuracy better than 10−3 absorbance units. Difference spectra of I− in chromatophores and in RCs closely correspond to each other. In the carbonyl stretching frequency region between 1640 and 1750 cm−1, bands are tentatively attributed to a shift (from 1713 to 1683 cm−1) of a keto carbonyl group, a change of an acetyl carbonyl grou at 1656 cm−1 and a decrease in absorbance strength of ester carbonyl groups (at 1746 and 1732 cm−P) after reduction of I. These groups likely belong to the BPh molecule, although at least one of the ester carbonyls could be assigned to an amino acid side chain. The absence of strong bands in the amide I and amide II region excludes large protein conformational changes associated with I reduction.