Using site-directed mutagenesis to probe the role of the D2 carotenoid in the secondary electron-transfer pathway of photosystem II

Abstract

Secondary electron transfer in photosystem II (PSII), which occurs when water oxidation is inhibited, involves redox-active carotenoids (Car), as well as chlorophylls (Chl), and cytochrome b559 (Cyt b559), and is believed to play a role in photoprotection. CarD2 may be the initial point of secondary electron transfer because it is the closest cofactor to both P680, the initial oxidant, and to Cyt b559, the terminal secondary electron donor within PSII. In order to characterize the role of CarD2 and to determine the effects of perturbing CarD2 on both the electron-transfer events and on the identity of the redox-active cofactors, it is necessary to vary the properties of CarD2 selectively without affecting the ten other Car per PSII. To this end, site-directed mutations around the binding pocket of CarD2 (D2-G47W, D2-G47F, and D2-T50F) have been generated in Synechocystis sp. PCC 6803. Characterization by near-IR and EPR spectroscopy provides the first experimental evidence that CarD2 is one of the redox-active carotenoids in PSII. There is a specific perturbation of the Car∙+ near-IR spectrum in all three mutated PSII samples, allowing the assignment of the spectral signature of CarD2∙+; CarD2∙+ exhibits a near-IR peak at 980 nm and is the predominant secondary donor oxidized in a charge separation at low temperature in ferricyanide-treated wild-type PSII. The yield of secondary donor radicals is substantially decreased in PSII complexes isolated from each mutant. In addition, the kinetics of radical formation are altered in the mutated PSII samples. These results are consistent with oxidation of CarD2 being the initial step in secondary electron transfer. Furthermore, normal light levels during mutant cell growth perturb the shape of the Chl∙+ near-IR absorption peak and generate a dark-stable radical observable in the EPR spectra, indicating a higher susceptibility to photodamage further linking the secondary electron-transfer pathway to photoprotection.Electronic supplementary materialThe online version of this article (doi:10.1007/s11120-013-9793-6) contains supplementary material, which is available to authorized users.

A Photosynthesis-Specific Rubredoxin-Like Protein Is Required for Efficient Association of the D1 and D2 Proteins during the Initial Steps of Photosystem II Assembly

“…The midpoint redox potential of RubA is 1125 mV (Wastl et al, 2000), which is positive enough to allow RubA to keep the acceptor side of PSII oxidized and so prevent the accumulation of singly reduced Q A (due to either inadvertent nonphotochemical reduction or lack of functional Q B ). Another possibility is that reduced RubA might reduce Cyt b 559 within PSII and so act as part of a photoprotective cyclic electron transport pathway involving the oxidation and subsequent rereduction of P680 1 via a pathway involving Q A , RubA, Cyt b 559 , and the Car D2 carotenoid bound to D2 (Shinopoulos et al, 2014). A redox role for RubA is supported by our observation that the DrubA strain is able to proliferate in autotrophic media under continuous illumination but not when exposed to fluctuating light consisting of cycles of 15 min of light and 15 min of dark ( Figure 4A).…”

Carotenoids Assist in Cyanobacterial Photosystem II Assembly and Function

“…Recently it has been shown that one of the two so-called redox active carotenoids, Car D2 , plays a role in photoprotection. Site-directed mutations around the binding pocket of Car D2 in Synechocystis cells revealed the importance of β-carotenes in the initiation of secondary electron transfer processes, which occurs when water oxidation is inhibited ( Shinopoulos et al, 2014 ).…”

Enhancing photosynthesis in plants: the light reactions

“…One can wonder if such success can be in part due to their superior photoprotective mechanisms and if any such traits can be engineered in crop plants to derive yield advantages under particular environmental conditions. It is worth pointing out that the exact molecular mechanism of cyclic electron flow around PSII is not completely understood, but is likely to involve electron transfer via Cyt b-559 and a carotenoid bound to the D2 subunit [ 62 ]. One obvious next step is to express the core subunits of PSII from these strains in a model plant.…”