Mechanisms of Photodamage and Protein Turnover in Photoinhibition.

Rapid protein degradation and replacement is an important response to photodamage and a means of photoprotection by recovering proteostasis. Protein turnover and translation efficiency studies have discovered fast turnover subunits in cytochrome b6f and the NAD(P)H dehydrogenase (NDH) complex, in addition to PSII subunit D1. Mutations of these complexes have been linked to enhanced photodamage at least partially via cyclic electron flow. Photodamage and photoprotection involving cytochrome b6f, NDH complex, cyclic electron flow, PSI, and nonphotochemical quenching proteins have been reported. Here, we propose that the rapid turnover of specific proteins in cytochrome b6f and the NDH complex need to be characterised and compared with the inhibition of PSII by excess excitation energy and PSI by excess electron flux to expand our understanding of photoinhibition mechanisms.

Conformational Changes in Pigment-Protein Complexes at Low Temperatures-Spectral Memory and a Possibility of Cooperative Effects.

We employed nonphotochemical hole burning (NPHB) and fluorescence line narrowing (FLN) spectroscopies to explore protein energy landscapes and energy transfer processes in dimeric Cytochrome b6f, containing one chlorophyll molecule per protein monomer. The parameters of the energy landscape barrier distributions quantitatively agree with those reported for other pigment-protein complexes involved in photosynthesis. Qualitatively, the distributions of barriers between protein substates involved in the light-induced conformational changes (i.e., -NPHB) are close to glass-like ∼1/√V (V is the barrier height) and not to Gaussian. There is a high degree of correlation between the heights of the barriers in the ground and excited states in individual pigment-protein systems, as well as nearly perfect spectral memory. Both NPHB and hole recovery are due to phonon-assisted tunneling associated with the increase of the energy of a scattered phonon. As the latter is unlikely for simultaneously both the hole burning and the hole recovery, proteins must exhibit a NPHB mechanism involving diffusion of the free volume toward the pigment. Entities involved in the light-induced conformational changes are characterized by md(2) value of about 1.0 × 10(-46) kg·m(2). Thus, these entities are protons or, alternatively, small groups of atoms experiencing sub-Å shifts. However, explaining all spectral hole burning and recovery data simultaneously, employing just one barrier distribution, requires a drastic decrease in the attempt frequency to about 100 MHz. This decrease may occur due to cooperative effects. Evidence is presented for excitation energy transfer between the chlorophyll molecules of the adjacent monomers. The magnitude of the dipole-dipole coupling deduced from the Δ-FLN spectra is in good agreement with the structural data, indicating that the explored protein was intact.

High-light induced singlet oxygen formation in cytochrome b(6)f complex from Bryopsis corticulans as detected by EPR spectroscopy.

Electron paramagnetic resonance (EPR) spectroscopy was used to detect the light-induced formation of singlet oxygen ((1)O(2)*) in the intact and the Rieske-depleted cytochrome b(6)f complexes (Cyt b(6)f) from Bryopsis corticulans, as well as in the isolated Rieske Fe-S protein. It is shown that, under white-light illumination and aerobic conditions, chlorophyll a (Chl a) bound in the intact Cyt b(6)f can be bleached by light-induced (1)O(2)*, and that the (1)O(2)* production can be promoted by D(2)O or scavenged by extraneous antioxidants such as l-histidine, ascorbate, beta-carotene and glutathione. Under similar experimental conditions, (1)O(2)* was also detected in the Rieske-depleted Cyt b(6)f complex, but not in the isolated Rieske Fe-S protein. The results prove that Chl a cofactor, rather than Rieske Fe-S protein, is the specific site of (1)O(2)* formation, a conclusion which draws further support from the generation of (1)O(2)* with selective excitation of Chl a using monocolor red light.

Stromal over-reduction by high-light stress as measured by decreases in P700 oxidation by far-red light and its physiological relevance.

The oxidation level of P700 induced by far-red light (DeltaA(FR)) in briefly dark-treated leaves of some sun plants decreased during the daytime and recovered at night. The dark recovery of decreased DeltaA(FR) proceeded slowly, with a half-time of about 5 h. We propose that stromal over-reduction induced by sunlight was the direct cause of the depression of DeltaA(FR). The depression of DeltaA(FR) found during the daytime was reproduced by controlled illumination with saturating light of fully dark-treated leaves. Simultaneous measurement of P700 redox and chlorophyll fluorescence showed that the depression of DeltaA(FR) was associated with dark reduction of the plastoquinone pool, which represented cyclic electron transport activity. The decrease of DeltaA(FR) in the light-stressed chloroplasts was partly reversed by treatment with 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, an inhibitor of electron transport at the cytochrome b6/f complex, and the subsequent addition of methyl viologen, an efficient electron acceptor from photosystem I (PSI), stimulated further recovery, showing that both cyclic electron flow around PSI and the charge recombination within PSI were responsible for the light-induced depression of DeltaA(FR). The dark level of blue-green fluorescence, an indicator of NAD(P)H concentration, from intact chloroplasts was increased by high-light stress, suggesting that NADPH accumulated in stroma as a result of the high-light treatment. Possible effects on photosynthetic activity of over-reduction and its physiological relevance are discussed.