Evidence for the protonation of two internal carboxylic groups during the photocycle of bacteriorhodopsin

Siebert, Friedrich; Mäntele, Werner; Kreutz, W.

doi:10.1016/0014-5793(82)80021-5

Cited by 146 publications

(99 citation statements)

References 33 publications

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“…The oxidase samples in 'Hz0 differed in concentration from that in 'HaO; the difference signal was thus normalized on the concentration of the 'Ha0 sample on the basis of the difference spectra in the visible spectral range. A shift of several wavenumbers (around 10 cm-') is observed upon 'H/'H exchange for the protonated side chains of Asp and Glu residues in model compounds [25], and was previously observed to be 4-10 cm-' for other proteins [26,27]. We take the spectral region and the observed shift as a conclusive argument to definitely attribute the signals to Asp or Glu COOH modes in the reduced (1733 cm-l) and the oxidized (1745 cm-l) state of the enzyme.…”

Section: Resultssupporting

confidence: 58%

Carboxyl group protonation upon reduction of the Paracoccus denitrificans cytochrome c oxidase: direct evidence by FTIR spectroscopy

et al. 1996

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The redox reactions of the cytochrome c oxidase from Puracoccus denitrificans were investigated in a thin-layer cell designed for the combination of electrochemistry under anaerobic conditions with UVNIS and IR spectroscopy. Quantitative and reversible electrochemical reactions were obtained at a surfacemodified electrode for all cofactors as indicated by the optical signals in the 400-700 nm range. Fourier transform infrared (FTIR) difference spectra of reduction and oxidation (reducedminus-oxidized and oxidized-minus-reduced, respectively) obtained in the 1800-1000 cm-' range reveal highly structured band features with major contributions in the amide 1(1620-1680 cm-') and amide II (1580-1520 cm-') range which indicate structural rearrangements in the cofactor vicinity. However, the small amplitude of the IR difference signals indicates that these conformational changes are small and affect only individual peptide groups. In the spectral region above 1700 cm-', a positive peak in the reduced state (1733 cm-') and negative peak in the oxidized state (1745 cm-') are characteristic for the formation and decay of a COOH mode upon reduction. The most obvious interpretation of this difference signal is proton uptake by one Asp or Glu side chain carboxyl group in the reduced state and deprotonation of another Asp or Glu residue. Moreover, both residues could well be coupled as a donor-acceptor pair in the proton transfer chain. An alternative interpretation is in terms of a protonated carboxyl group which shifts to a different environment in the reduced state. The relevance of this first direct observation of protein protonation changes in the cytochrome c oxidase for vectorial proton transfer and the catalytic reaction is discussed.

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Section: Resultssupporting

confidence: 58%

Carboxyl group protonation upon reduction of the Paracoccus denitrificans cytochrome c oxidase: direct evidence by FTIR spectroscopy

et al. 1996

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“…Since IR difference spectroscopy, as applied here, is not selective to Chl molecular vibrations, carbonyl absorption changes from amino acid side chains, the peptide backbone (as discussed above) and also lipid esters have to be taken into account. Protonation of individual carboxyl groups in bacteriorhodopsin, for example, gives rise to IR difference bands [12] comparable in position and magnitude to the 1750 cm-' (1755 cm-') band. Exchange of 'Hz0 against 'Hz0 performed through the vapor phase in the hydration cell for 24 h, however, did not change the 1750 cm-' (1755 cm-') band position in the P+ spectra (a downward shift of approx.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 1985

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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 1800450 cm-l 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 = 0 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. Fourier transform infrared spectroscopyReaction center Bacterial photosynthesis Primary donor Chlorophyll

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“…Caged ATP is an inactive, photolabile ATPderivative that releases ATP upon ultraviolet illumination [ 15,161. This 'hands-off' photochemically triggered difference spectroscopy avoids uncertainties due to buffer subtraction or different sample concentration and thus allows a comparison of different enzyme states on the level of individual bonds, as has been demonstrated before for photobiological [17,18] [22] by partial drying of a SR vesicle suspension on a CaFz IR window and sealing of the sample with a second window separated by a 6 pm spacer. 'Normal' ATPase samples contained 100-150 pg protein, 300 nmol 4-morpholinopropanesulphonic acid (Mops)/Tris (pH 6.8), 150 nmol KCI, 12 nmol MgC12, 0.12 nmol CaC12 in addition to the Ca* + bound by SR, 15 nmol caged ATP, 0.1 nmol Ca*+-ionophore A23187 and 10 nmol glutathione.…”

Section: Introductionmentioning

confidence: 99%

Molecular changes in the sarcoplasmic reticulum calcium ATPase during catalytic activity

1990

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Fourier transform infrared spectroscopy was used to study ligand binding and conformational changes in the CaZ+-ATPase of sarcoplasmic reticulum. Novel in infrared difference spectroscopy, the catalytic cycle in the IR sample was started by photolytic release of ATP from an inactive, photolabile ATP-derivative (caged ATP). Small, but characteristic infrared absorbance changes were observed upon ATP release. On the basis of model spectra, the absorbance changes corresponding to the trigger and substrate reactions, i.e. to photolysis of caged ATP and hydrolysis of ATP, were separated from the absorbance changes due to the active ATPase reflecting formation of the phosphorylated Ca$,P enzyme form.A major rearrangement of ATPase conformation as the result of catalysis can be excluded.

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Evidence for the protonation of two internal carboxylic groups during the photocycle of bacteriorhodopsin

Cited by 146 publications

References 33 publications

Carboxyl group protonation upon reduction of the Paracoccus denitrificans cytochrome c oxidase: direct evidence by FTIR spectroscopy

Carboxyl group protonation upon reduction of the Paracoccus denitrificans cytochrome c oxidase: direct evidence by FTIR spectroscopy

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

Molecular changes in the sarcoplasmic reticulum calcium ATPase during catalytic activity

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