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In bacterial reaction centers the charge separation process across the photosynthetic membrane is predominantly driven by the excited state of the bacteriochlorophyll dimer (D). An X-ray structure analysis of the Phe M197→Tyr mutant reaction center from Rhodobacter sphaeroides at 2.7 A resolution suggests the formation of a hydrogen bond as postulated by Wachtveitl et al. [Biochemistry 32, 12875–12886, 1993] between the Tyr M197 hydroxy group and one of the 2a-acetyl carbonyls of D. In combination with electrochemically induced FTIR difference spectra showing a split band of the π-conjugated 9-keto carbonyl of D, there is clear evidence for the existence of such a hydrogen bond.

The binding and interaction of carbamoylcholine with the nicotinic acetylcholine receptor was investigated using photolytically release carbamoylcholine (‘caged’ carbamoylcholine). Upon UV flash activation of this photolabile substrate analog, characteristic changes in the IR absorbance spectrum were detected. Apart from difference bands arising from the changes of molecular structure upon photolytical release, spectral features can be attributed to the agonist upon binding to the receptor as well as to conformational changes of the receptor itself. The two photo-labile agonist analogs N -[1-(2-nitrophenyl)ethyl] carbamoylcholine iodide (cage I) and N -(α-carboxy-2-nitrobenzyl) carbamoylcholine trifluoroacetate (cage II), with different structures for comparison of the 1680-1540 cm −1 region sensitive for protein conformation, yielded consistent results. A preliminary interpretation in terms of substrate binding and local conformational changes of the receptor upon carbamoylcholine binding is provided, in analogy to the binding of acetylcholine, activation, and subsequent deactivation taking place during signal transduction.

The redox and spectral characteristics of the 4-heme cytochrome c unit of the photochemical reaction center from Rhodopseudomonas viridis were studied by a combination of protein electrochemistry and spectroscopy using an ultra thin-layer spectroelectrochemical cell. Quantitative and reversible reduction of the high-potential and the low-potential hemes was performed in cyclic titrations to record the optical difference spectra in the α-band region. The titration of the absorbance from the high-potential hemes can be approximated with a sum of 2 Nernst functions with Em = 0.113 V and Em = 0.175 V. The corresponding titration of the absorbance from the low-potential hemes yielded Em = −0.257 V and Em = −0.175 V (all potentials quoted vs. Ag/AgCl/3 M KCl; add 0.208 V for potentials vs. standard hydrogen electrode). The high-potential hemes equilibrate rapidly and titrate identically for oxidative and reductive titrations. Under identical conditions, the low-potential hemes exhibit a hysteresis, thus indicating much slower equilibration with the applied potential. Cyclic titrations with increasing equilibration periods, however, indicate the disappearance of the hysteresis for equilibration periods approximately twice as long as for the high-potential hemes. We take this as evidence for a slower internal equilibration, but against a cooperativity of the low-potential hemes as observed for other multi-heme cytochromes.

Lipid protein interactions play a key role in the stability and function of various membrane proteins. Earlier we have reported the extreme thermal stability of porin from Paracoccus denitrificans reconstituted into liposomes. Here, we used Fourier transform infrared spectroscopy for a label free analysis of the global secondary structural changes and local changes in the tyrosine microenvironment. Our results show that a mixed lipid system (non-uniform bilayer) optimizes the thermal stability of porin as compared to the porin in pure lipids (uniform bilayer) or detergent micelles. This is in line with the fact that the bacterial outer membrane is a dynamic system made up of lipids of varying chain lengths, head groups and the barrel wall height contacting the membrane is uneven.

The redox dependent changes in the cytochrome c oxidase from bovine heart were studied with a combined electrochemical and FT-IR spectroscopic approach. A direct comparison to the electrochemically induced FT-IR difference spectra of the cytochrome c oxidase from Paracoccus denitrificans reveals differences in the structure and intensity of vibrational modes. These differences are partially attributed to interactions of subunits influencing the heme and protein modes. In the spectral regions characteristic for ν(C=O) and ν(COO−)s/as modes of protonated and deprotonated Asp and Glu residues, additional signals at 1736, 1602 and 1588 cm−1 are observed. On this basis, the possible involvement of Asp-51, a residue specifically conserved in mammalian oxidase and previously proposed to show redox depended conformational changes in the respective X-ray structures, is critically discussed.

Fourier transform infrared spectroscopy was used to study ligand binding and conformational changes in the Ca2+-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 Ca2E1P enzyme form. A major rearrangement of ATPase conformation as the result of catalysis can be excluded.

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 1630 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 15601550 cm−1.

A pigment-protein complex containing a reaction center and the B875 light-harvesting complex from Rhodopseudomonas palustris was purified and crystallized in the presence of detergent. Thin, rectangular crystals were obtained and used for optical spectroscopy. The protein pattern as well as the spectrum of the solubilized and crystallized complex show that the purified complex was crystallized without a major change in its composition and conformation. The absorption band intensities in the plane of the crystal as well as the spectra of the tilted crystal indicate that the 590 nm Q x transitions are aligned along the long axis of the crystal, while the 875 nm Q y transitions are aligned along the short axis of the crystal plane. The results favor a model where the antenna bacteriochlorophylls associated with one reaction center have a common Q x direction and perpendicular to it a distribution of 875 nm Q y transitions exhibiting a maximum along one direction.

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.

A comparison is made between the PQ A → P + Q − A and PQ A Q B → P + Q A Q − 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 + Q A -minus-PQ A spectrum and not in the P + Q A Q − B -minus-PQ A Q B spectrum. In contrast, this signal is present in both P + Q − A -minus-PQ − A and P + Q A Q − B -minus-PQ A Q B spectra of Rps. viridis RCs. These data are interpreted in terms of a conformational change of the protein backbone near Q A (possible at the peptide CO of a conserved alanine residue in the Q A pocket) and of the different bonding interactions of Q B with the protein in the RC of the two species.

Thin three-dimensional crystals of the light-harvesting chlorophyll a/b-protein complex from pea chloroplast membranes were investigated by microspectrophotometry. Measurements of tilted crystals revealed pronounced linear dichroism in a direction perpendicular to the crystal plane but no observable dichroism in the crystal plane. Qy transition moments of chlorophylls were rotationally isotropic with respect to the crystal plane and partially aligned in this plane. Qx transition moments showed no preferential orientation and appeared to be wholly isotropic. The orientation of transition moments in the crystal was consistent with three-fold symmetry of the light-harvesting complex indicated by the crystal structure. The linear dichroism of the crystals reflected the optical properties of the complex in the photosynthetic membrane, indicating a highly symmetrical arrangement of antenna chlorophylls.

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.

The redox reactions of the cytochrome c oxidase from Paracoccus denitrificans were investigated in a thin-layer cell designed for the combination of electrochemistry under anaerobic conditions with UV/VIS and IR spectroscopy. Quantitative and reversible electrochemical reactions were obtained at a surface-modified 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 (reduced-minus-oxidized and oxidized-minus-reduced, respectively) obtained in the 1800-1000 cm(-1) range reveal highly structured band features with major contributions in the amide I (1620-1680 cm(-1)) and amide II (1580-1520 cm(-1)) 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(-1), a positive peak in the reduced state (1733 cm(-1)) and negative peak in the oxidized st ate (1745 cm(-1)) 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.

Thin three-dimensional crystals of the photochemical reaction center from Rhodobacter sphaeroides wild-type strain Y were investigated with microspectrophotometric methods. Absorbance and linear dichroism spectra were obtained for two crystal forms with the same type of unit cell, but grown in different morphological forms. High dichroism was found for the Q x and the Q y transitions of the bacteriopheophytin as well as for the Q y transitions of the accessory bacteriochlorophylls and the bacteriochlorophylls forming the primary electron donor. Low absorption and dichroism of the carotenoid transitions in the plane of the crystal suggests a large component out of the crystal plane. Photoactivity of the crystal was demonstrated by light-induced charge separation. The analysis of the rates of charge recombination indicates that almost all reaction centers in the crystal (> 90%) contain the secondary electron acceptor Q B .