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We have developed a new technique for the study of redox-linked conformational changes in proteins, by the combination of two established techniques. Fourier-transform infrared spectroscopy has been used together with direct electrochemistry of the protein at a modified metal electrode surfke. The technique has been evaluated with cytochrome c, because of its well-characterized electrochemistry and because the availability of X-ray crystallographic and NMR studies of both redox states of the protein provides a reference against which our data can be compared. In electrochemical control experiments, it was confirmed that the spectroelectrochemical cell design allows fast, accurate and reproducible control of the redox poise of the protein. The resulting reducedminus-oxidized infrared difference spectra show the changes in the frequencies and intensities of molecular vibrations which arise from the redox-linked conformational change. In contrast to the absolute infrared spectra of proteins, such difference spectra can be sufficiently straightforward to allow interpretation at the level of individual bonds. A complete interpretation of the spectra is beyond the scope of the present paper: however, on the basis of the data presented, we are able to suggest assignments for all except one of the major bands between 1500 cm-' and 1800 cm-'.Infrared spectroscopy is the classical method for structural and functional investigations of small molecules, having the advantage that the spectrum is a function of the entire molecular structure, rather than just that of a chromophore. However, when applied to proteins this becomes a disadvantage, since the very large number of overlapping signals gives rise to a spectrum consisting of only a few broad bands. Such spectra can yield valuable information at the level of secondary structure [l, 21, but interpretation at the level of individual bonds is not usually feasible; for this reason X-ray crystallography and, more recently, NMR spectroscopy are the preferred methods in studies of protein structure. Nevertheless, it is clear that the ultimate goal of structural investigations of proteins, a detailed understanding of the mechanisms of their reactions, is most likely to be achieved through information obtained by a number of complementary techniques. Furthermore, infrared spectroscopy has been used to obtain information on the structures of high-molecular-mass proteins, in solution and membrane-bound, which is not obtainable by either of the above methods. Thus extraction of detailed information from infrared spectra of proteins remains a desirable goal.One very useful strategy has been differential spectroscopy, in which the spectra of a protein in two different states are obtained. Subtraction yields a difference spectrum arising from a particular structural change in the protein, with the [5]. However, in all of these cases the signal studied was in a spectral region where the background absorption due to the protein and the solvent was low, thus allowing sufficiently accurat...

Despite the apparent similarity between the plant Photosystem II reaction center (RC) and its purple bacterial counterpart, we show in this work that the mechanism of charge separation is very different for the two photosynthetic RCs. By using femtosecond visible-pump-mid-infrared probe spectroscopy in the region of the chlorophyll ester and keto modes, between 1,775 and 1,585 cm ؊1 , with 150-fs time resolution, we show that the reduction of pheophytin occurs on a 0.6-to 0.8-ps time scale, whereas P ؉ , the precursor state for water oxidation, is formed after Ϸ6 ps. We conclude therefore that in the Photosystem II RC the primary charge separation occurs between the ''accessory chlorophyll'' ChlD1 and the pheophytin on the so-called active branch.electron transfer ͉ photosynthesis ͉ pump-probe T he primary steps of energy and electron transfer in green plants' photosynthesis occur in two large protein complexes called Photosystem I and Photosystem II (PSII). PSII is an aggregate of many individual pigment-protein complexes. The core of PSII consists of the chlorophyll (Chl)-binding antennaproteins CP43 and CP47, which feed excitation energy into the D 1 D 2 cytb559 reaction center (RC). Crystal structures of PSII cores from cyanobacteria have been resolved with increasingly high resolution (1-3); currently, the resolution is 3.2 Å (4). The structure of the PSII RC shows four Chls and two pheophytins (H) arranged in two branches very similar to the bacterial RC. In the heart of the PSII RC, there is a dimer of Chls, and in each branch there is one monomeric Chl and one H. Furthermore, there are two distant Chls bound to the periphery of the PSII RC. Although the structure suggests there may be a ''special pair'' of strongly electronically coupled pigments in the PSII RC, the visible absorption spectrum does not show a distinct band. This finding is in contrast to the bacterial RC, where the lowest energy absorption band fully originates from one of the exciton transitions of a special pair of bacteriochlorophylls.Since the first purification of the PSII RC in 1987 (5), it has been speculated that its way of operation would be similar to that of the bacterial RC, with a special pair that upon excitation drives a charge separation in Ϸ3 ps. This idea was based on the strong homology between the bacterial RC and the PSII RC, the strong similarity in the pigment composition, even details in the way the pigments interacted with the protein, and the near-identity of the electron transfer events at the acceptor side. Conversely, it was clear that major differences between the two RCs had to exist at the electron donor side where in the PSII RC charge separation eventually leads to the oxidation of water and the production of molecular oxygen, requiring a very large oxidation potential of the primary electron donor (Ͼ1.2 V vs. 0.45 V in the bacterial RC).In the mid-1990s, it was recognized that energy transfer and charge separation in the PSII RC most likely proceeded in a manner that is very different from that in the b...

We have characterized a xanthophyll binding site, called V1, in the major light harvesting complex of photosystem II, distinct from the three tightly binding sites previously described as L1, L2, and N1. Xanthophyll binding to the V1 site can be preserved upon solubilization of the chloroplast membranes with the mild detergent dodecyl-␣-D-maltoside, while an IEF purification step completely removes the ligand. Surprisingly, spectroscopic analysis showed that when bound in this site, xanthophylls are unable to transfer absorbed light energy to chlorophyll a. Pigments bound to sites L1, L2, and N1, in contrast, readily transfer energy to chlorophyll a. This result suggests that this binding site is not directly involved in light harvesting function. When violaxanthin, which in normal conditions is the main carotenoid in this site, is depleted by the de-epoxidation in strong light, the site binds other xanthophyll species, including newly synthesized zeaxanthin, which does not induce detectable changes in the properties of the complex. It is proposed that this xanthophyll binding site represents a reservoir of readily available violaxanthin for the operation of the xanthophyll cycle in excess light conditions.Light energy for higher plant photosynthesis is harvested by pigments including chlorophyll (Chl) 1 a, Chl b, and carotenoids bound to pigment binding proteins embedded into the thylakoid membrane. Each photosystem is made of two moieties: the core complex, containing Chl a and ␤-carotene bound to plastidencoded polypeptides, and the light harvesting system, made up of nuclear encoded proteins of the Lhc family which, besides Chl a, also bind Chl b and the three xanthophylls lutein, violaxanthin, and neoxanthin. LHCII is by far the major antenna complex, since it binds about 50% of total chlorophyll. It is composed of heterotrimeric complexes of the Lhcb1, -2, and -3 gene products (1). Electron crystallography and mutation analysis showed that each Lhcb1 subunit contains five Chl a and four Chl b binding sites, while three additional sites can bind either Chl a or Chl b. Chlorophyll ligands are amino acid side chains belonging to three trans-membrane ␣-helices or to neighbor Chl (2, 3) through coordination of the Mg 2ϩ atom at the center of each porphyrin. Within the pigment-protein complex are also located two carotenoid binding sites, called L1 and L2, cross-bracing helices A and B. These sites are occupied by lutein (L1) and by lutein (80%) and violaxanthin (20%) (L2) (4, 5). A third carotenoid binding site (N1), highly specific for neoxanthin, was localized in the C helix domain (6). Xanthophylls in sites L1, L2, and N1, are tightly bound to the complex even in very harsh conditions of purification. In addition, violaxanthin can be bound to LHCII when isolated by mild detergent treatment, while the interaction does not survive further purification steps, such as IEF, suggesting loose binding (7,8).Besides their role in light harvesting, xanthophylls are also involved in photoprotection by quenching 3 Chl...

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)

The effect of global (15)N or (2)H labeling on the light-induced P700(+)/P700 FTIR difference spectra has been investigated in photosystem I samples from Synechocystis at 90 K. The small isotope-induced frequency shifts of the carbonyl modes observed in the P700(+)/P700 spectra are compared to those of isolated chlorophyll a. This comparison shows that bands at 1749 and 1733 cm(-)(1) and at 1697 and 1637 cm(-)(1), which upshift upon formation of P700(+), are candidates for the 10a-ester and 9-keto C=O groups of P700, respectively. A broad and relatively weak band peaking at 3300 cm(-)(1), which does not shift upon global labeling or (1)H-(2)H exchange, is ascribed to an electronic transition of P700(+), indicating that at least two chlorophyll a molecules (denoted P(1) and P(2)) participate in P700(+). Comparisons of the (3)P700/P700 FTIR difference spectrum at 90 K with spectra of triplet formation in isolated chlorophyll a or in RCs from photosystem II or purple bacteria identify the bands at 1733 and 1637 cm(-)(1), which downshift upon formation of (3)P700, as the 10a-ester and 9-keto C=O modes, respectively, of the half of P700 that bears the triplet (P(1)). Thus, while the P(2) carbonyls are free from interaction, both the 10a-ester and the 9-keto C=O of P(1) are hydrogen bonded and the latter group is drastically perturbed compared to chlorophyll a in solution. The Mg atoms of P(1) and P(2) appear to be five-coordinated. No localization of the triplet on the P(2) half of P700 is observed in the temperature range of 90-200 K. Upon P700 photooxidation, the 9-keto C=O bands of P(1) and P(2) upshift by almost the same amount, giving rise to the 1656(+)/1637(-) and 1717(+)/1697(-) cm(-)(1) differential signals, respectively. The relative amplitudes of these differential signals, as well as of those of the 10a-ester C=O modes, appear to be slightly dependent on sample orientation and temperature and on the organism used to generate the P700(+)/P700 spectrum. If it is assumed that the charge density on ring V of chlorophyll a, as measured by the perturbation of the 10a-ester or 9-keto C=O IR vibrations, mainly reflects the spin density on the two halves of the oxidized P700 special pair, a charge distribution ranging from 1:1 to 2:1 (in favor of P(2)) is deduced from the measurements presented here. The extreme downshift of the 9-keto C=O group of P(1), indicative of an unusually strong hydrogen bond, is discussed in relation with the models previously proposed for the PSI special pair.

Photosystem I of higher plants is characterized by a typically long wavelength fluorescence emission associated to its light-harvesting complex I moiety. The origin of these low energy chlorophyll spectral forms was investigated by using site-directed mutagenesis of Lhca1-4 genes and in vitro reconstitution into recombinant pigment-protein complexes. We showed that the red-shifted absorption originates from chlorophyll-chlorophyll (Chl) excitonic interactions involving Chl A5 in each of the four Lhca antenna complexes. An essential requirement for the presence of the red-shifted absorption/fluorescence spectral forms was the presence of asparagine as a ligand for the Chl a chromophore in the binding site A5 of Lhca complexes. In Lhca3 and Lhca4, which exhibit the most red-shifted red forms, its substitution by histidine maintains the pigment binding and, yet, the red spectral forms are abolished. Conversely, in Lhca1, having very low amplitude of red forms, the substitution of Asn for His produces a red shift of the fluorescence emission, thus confirming that the nature of the Chl A5 ligand determines the correct organization of chromophores leading to the excitonic interaction responsible for the red-most forms. The red-shifted fluorescence emission at 730 nm is here proposed to originate from an absorption band at ϳ700 nm, which represents the low energy contribution of an excitonic interaction having the high energy band at 683 nm. Because the mutation does not affect Chl A5 orientation, we suggest that coordination by Asn of Chl A5 holds it at the correct distance with Chl B5.Photosystem I is a multisubunit pigment-protein complex of the chloroplast membrane acting as a plastocyanin/ferredoxin oxido-reductase in oxygenic photosynthesis. One important spectroscopic feature of PSI 1 is the presence of Chls absorbing at energy lower than the PSI primary electron donor, P700.Although these spectral forms account for only a small percentage of the total absorption, their effect in the energy transfer and trapping of PSI is very prominent (1), with at least 80% of excitation in the complex transiting through them on their way to P700 (2). It has been widely proposed that these forms represent the low energy contributions of excitonic interactions, which involve two or more Chl molecules (3-5); however, the identity of the chromophores involved and the details of the interaction are still unknown.Although the presence of low energy-absorbing Chls is ubiquitous in the PSI of different organisms, their amounts and energies appear to be highly species-dependent (1). In the PSI of higher plants, the red forms are associated with the outer antenna, LHCI (6, 7). LHCI is composed by four pigmentbinding proteins, namely Lhca1-4 (6, 8). These complexes, localized on one side of the core complex (9, 10), are organized in dimers with 10 Chl molecules per subunit (11).As for their properties, Lhca2 and Lhca4 differ from Lhca1 and Lhca3 in many respects. The first two have higher Chl b content with respect to Lhca1 and Lhca3 an...