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

AbstractFourier 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

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

AbstractA 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

Coupling of electron transfer to proton uptake at the QB site of the bacterial reaction center : A perspective from FTIR difference spectroscopy

AbstractFTIR difference spectroscopy provides a unique approach to study directly protonation/deprotonation events of carboxylic acids involved in the photochemical cycle of membrane proteins, such as the bacterial photosynthetic reaction center (RC). In this work, we review the data obtained by light-induced FTIR difference spectroscopy on the first electron transfer to the secondary quinone QB in native RCs and a series of mutant RCs. We first examine the approach of isotope-edited FTIR spectroscopy to investigate the binding site of QB. This method provides highly specific IR vibrational fingerprints of the bonding interactions of the carbonyls of QB and QB− with the protein. The same isotope-edited IR fingerprints for the carbonyls of neutral QB have been observed for native Rhodobacter sphaeroides RCs and several mutant RCs at the Pro-L209, Ala-M260, or Glu-L212/Asp-L213 sites, for which X-ray crystallography has found the quinone in the proximal position. It is concluded that at room temperature QB occupies a single binding site that fits well the description of the proximal site derived from X-ray crystallography and that the conformational gate limiting the rate of the first electron transfer from QA−QB to QAQB− cannot be the movement of QB from its distal to proximal site. Possible alternative gating mechanisms are discussed. In a second part, we review the contribution of the various experimental measurements, theoretical calculations, and molecular dynamics simulations which have been actively conducted to propose which amino acid side chains near QB could be proton donors/acceptors. Further, we show how FTIR spectroscopy of mutant RCs has directly allowed several carboxylic acids involved in proton uptake upon first electron transfer to QB to be identified. Owing to the importance of a number of residues for high efficiency of coupled electron transfer reactions, the photoreduction of QB was studied in a series of single mutant RCs at Asp-L213, Asp-L210, Asp-M17, Glu-L212, Glu-H173, as well as combinations of these mutations in double and triple mutant RCs. The same protonation pattern was observed in the 1760–1700 cm−1 region of the QB−/QB spectra of native and several mutant (DN-L213, DN-L210, DN-M17, EQ-H173) RCs. However, it was drastically modified in spectra of mutants lacking Glu at L212. The main conclusion of this work is that in native RCs from Rb. sphaeroides, Glu-L212 is the only carboxylic acid residue that contributes to proton uptake at all pH values (from pH 4 to pH 11) in response to the formation of QB−. Another important result is that the residues Asp-L213, Asp-L210, Asp-M17, and Glu-H173 are mostly ionized in the QB state at neutral pH and do not significantly change their protonation state upon QB− formation. In contrast, interchanging Asp and Glu at L212 and L213 (i.e., in the so-called swap mutant) led to the identification of a novel protonation pattern of carboxylic acids: at least four individual carboxylic acids were affected by QB reduction. The pH dependence of IR carboxylic signals in the swap mutant demonstrates that protonation of Glu-L213 occurred at pH >5 whereas that of Asp-L212 occurred over the entire pH range from 8 to 4. In native RCs from Rhodobacter sphaeroides, a broad positive IR continuum around 2600 cm−1 in the QB−/QB steady-state FTIR spectrum in 1H2O was assigned to delocalized proton(s) in a highly polarizable hydrogen-bonded network. The possible relation of the IR continuum band to the carboxylic acid residues and to bound water molecules involved in the proton transfer pathway was investigated by testing the robustness of this band to different mutations of acids. The presence of the band is not correlated with the localization of the proton on Glu-L212. The largest changes of the IR continuum were observed in single and double mutant RCs where Asp-L213 is not present. It is proposed that the changes observed in the mutant RCs with respect to native RCs reflect the specific role of bound protonated water molecule(s) located in the vicinity of Asp-L213 and undergoing hydrogen-bond changes in the network

The orientation of beta-sheets in porin. A polarized Fourier transform infrared spectroscopic investigation.

The orientation of the protein secondary structures in porin is investigated by Fourier transform infrared (FTIR) linear dichroism of oriented multilayers of porin reconstituted in lipid vesicles. The FTIR absorbance spectrum shows the amide I band at 1,631 cm-1 and several shoulders around 1,675 cm-1 and at 1,696 cm-1 indicative of antiparallel beta-sheets. The amide II is centered around 1,530 cm-1. The main dichroic signals peak at 1,738, 1,698, 1,660, 1,634, and 1,531 cm-1. The small magnitude of the 1,634 cm-1 and 1,531 cm-1 positive dichroism bands demonstrates that the transition moments of the amide I and amide II vibrations are on the average tilted at 47 degrees +/- 3 degrees from the membrane normal. This indicates that the plane of the beta-sheets is approximately perpendicular to the bilayer. From these IR dichroism results and previously reported diffuse x-ray data which revealed that a substantial number of beta-strands are nearly perpendicular to the membrane, a model for the packing of beta-strands in porin is proposed which satisfies both IR and x-ray requirements. In this model, the porin monomer consists of at least two beta-sheet domains, both with their plane perpendicular to the membrane. One sheet has its strands direction lying nearly parallel to the membrane normal while the other sheet has its strands inclined at a small angle away from the membrane plane