Javier De Las Rivas

Two sites of primary degradation of the D1-protein induced by acceptor or donor side photo-inhibition in photosystem II core complexes

Depending on experimental conditions we have found that photo‐inhibitory treatment of photosystem II (PSII) core complexes, isolated from wheat, can generate two fragments of about 23–24 kDa that contain either the C‐terminal or N‐terminal regions of the D1‐protein. A 24 kDa C‐terminal fragment appears when the water splitting reaction is not functional and an electron acceptor is present. This ‘donor’‐side inhibition also generates an N‐terminal fragment of about 10 kDa and is suggested to be due to the cleavage of a peptide bond in the region connecting transmembrane segments I and II of the D1‐protein. In contrast, an N‐terminal 23 kDa D1‐protein fragment is detected when the water splitting reactions of the isolated complex are active, and occurs in the absence of an added electron acceptor. This ‘acceptor’‐side photo‐inhibition also generates a C‐terminal fragment of about 10 kDa.

Two coupled β-carotene molecules protect P680 from photodamage in isolated Photosystem II reaction centres

Abstract The illumination of isolated Photosystem II (PS II) reaction centres in the presence of the artificial electron acceptors, silicomolybdate (SiMo) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) leads to the irreversible bleaching of β-carotene. The bleaching is biphasic with approx. 50% of the carotenoid being bleached rapidly and the remainder more slowly. This is attributed to the sequential bleaching of the two β-carotenes contained in the isolated complex, due to oxidation by the primary oxidant, P680+. Using SiMo as electron acceptor we found that only when all the β-carotene is irreversibly bleached does further illumination induce a loss of electron transfer activity. This rate of loss is exacerbated by the presence of oxygen. In addition to β-carotene degradation there is also, under the same conditions, an irreversible bleaching of chlorophyll absorbing at 670 nm. It is proposed therefore that β-carotene and chlorophyll 670 contained in the reaction centre protect against deleterious effects due to the photoaccumulation of the highly oxidising P680+ state by acting as electron donors. Comparison of the spectra of the normal 2 β-carotene and a 1 β-carotene containing preparation of the PS II reaction centre indicates that in the former these two carotenoids are excitonically coupled. The excitonic coupling allows a more rapid initial rate of electron donation by the β-carotene to P680+, than is seen in the preparation with only one carotenoid.

Three-dimensional electron cryo-microscopy study of the extrinsic domains of the oxygen-evolving complex of spinach: Assignment of the PsbO protein

Three independent three-dimensional reconstructions of the spinach photosystem II-light-harvesting complex supercomplex were derived from single particle analyses of non-stained, vitrified samples imaged by electron microscopy. Each reconstruction was found to differ significantly in the composition of the lumenal oxygen-evolving complex extrinsic proteins. From difference mapping, aided by electron microscopy of negatively stained selectively washed samples, regions of density were assigned to the PsbO and PsbP/PsbQ proteins. Interpretation of the density assigned to the PsbO protein was explored using computer-aided structural predictions. PsbO is calculated to be mainly a β-protein (38% β) composed of two domains within an overall elongated shape (Pazos, F., Heredia, P., Valencia, A., and De Las Rivas, J. (2001) Proteins Struct. Funct. Genet. 45, 372–381). The positioning and fitting of the proposed structural model for the PsbO protein within the three-dimensional map indicated that there is a single copy per reaction center. Moreover, the structural model derived for PsbO, together with difference mapping, indicates that this protein stretches across the surface of the reaction center with its N- and C-terminal domains located toward the CP47 and CP43 side, respectively. This structural assignment is discussed in terms of the recent x-ray-derived cyanobacterial model of PSII (Zouni, A., Witt, H.-T., Kern, J., Fromme, P., Krauss, N., Saenger, W., and Orth, P. (2001) Nature 409, 739–743).

Effect of Photosystem II inhibitor K-15 on photochemical reactions of the isolated D1/D2 cytochrome b559 complex

Effect of a highly efficient inhibitor of Photosystem II (PS II), K-15 (4-[methoxy-bis-(trifluoromethyl)methyl)-2,6-dinitrophenyl hydrazone methyl ketone), was investigated using the D1/D2/cytochrome b559 reaction centre (RC) complex. A novel approach for photoaccumulating reduced pheophytin (Pheo−) in the absence of the strong reducing agent, sodium dithionite, was demonstrated which involved illumination in the presence of TMPD (from 5 to 100 μM) under anaerobic conditions. The addition of K-15 at concentrations of 0.5 μM and 2 μM resulted in approx. 50% and near 100%, respectively, inhibition of this photoreaction, while subsequent additions of dithionite eliminated the inhibitory effect of K-15. Methyl viologen induced similar inhibition at much higher concentrations (>1 mM). Moreover, K-15 efficiently quenched the ‘variable’ part of chlorophyll fluorescence (which is the recombination luminescence of the pair P680+ Pheo−). A 50% inhibition was induced by 5 μM K-15 and the effect was maximal in the range 20 to 200 μM. Photooxidation of P680 in the presence of 0.1 mM silicomolybdate was also efficiently inhibited by K-15 (50% inhibition at 15 μM). The data are consistent with the idea put forward earlier (Klimov et al. 1992) that the inhibitory effect of K-15 is based on facilitating a rapid recombination between Pheo− and P680+ (or Z+) via its redox properties. The inhibitor can be useful for suppressing PS II reactions in isolated RCs of PS II which are resistant to all traditional inhibitors, like diuron, and probably functions by substituting for QA missing in the preparation.At a concentration of 0.5–50 μM K-15 considerably increased both the rate and extent of cytochrome b559 photoreduction in the presence, as well as in the absence, of 5 mM MnCl2. Consequently it is suggested that K-15 also serves as a mediator for electron transfer from Pheo− to cytochrome b559.

A comparison of the photochemical activity of two forms of Photosystem II reaction centre isolated from sugar beet

Abstract Both time-resolved fluorescence and absorption measurements have been conducted on two different forms of Photosystem II reaction centre isolated from sugar beet. One form, called RC IIa, contained 6 chlorophylls and 2 β-carotenes per 2 pheophytins, while the other, called RC Iib, contained 4 chlorophylls and 1 β-carotene per 2 pheophytins. Single photon-counting fluorescence decay obtained from the two preparations showed similar charge recombination fluorescence lifetimes which could be resolved into two components of 46.1 and 14.2 ns. Analysis of the amplitude of the fluorescence of the fast component of 5.6 ns, which largely originates from non-functional chlorophyll, gave an estimate of the relative activity for RC IIb which was only a 5.5% lower compared to that of RC IIa. This small relative difference in photochemical activity was also confirmed by measuring the extent of primary charge separation activity using flash induced absorption spectroscopy. In this case the amplitude of the long-lived component, attributed to primary radical-pair formation and recombination, was 16% lower in RC IIb as compared with RC IIa. When the secondary electron transfer activity of the two forms of reaction centre were measured using MnCl 2 and silicomolibdate as electron donor and acceptor respectively, RC IIb was 16% less active than RC IIa. From the data we conclude that the removal of 2 chlorophylls and 1 β-carotene molecules from the isolated Photosystem II reaction centre only slightly impair its functional activity with respect to primary charge separation. This conclusion seems to suggest that photochemically active isolated reaction centres of Photosystem II and purple bacteria can have the same minimum pigment stoichiometry of 4 chlorophylls and 1 carotenoid per 2 pheophytins.