Dataset Information

The Roles of Cytochrome b 559 in Assembly and Photoprotection of Photosystem II Revealed by Site-Directed Mutagenesis Studies.

ABSTRACT: Cytochrome b 559 (Cyt b 559) is one of the essential components of the Photosystem II reaction center (PSII). Despite recent accomplishments in understanding the structure and function of PSII, the exact physiological function of Cyt b 559 remains unclear. Cyt b 559 is not involved in the primary electron transfer pathway in PSII but may participate in secondary electron transfer pathways that protect PSII against photoinhibition. Site-directed mutagenesis studies combined with spectroscopic and functional analysis have been used to characterize Cyt b 559 mutant strains and their mutant PSII complex in higher plants, green algae, and cyanobacteria. These integrated studies have provided important in vivo evidence for possible physiological roles of Cyt b 559 in the assembly and stability of PSII, protecting PSII against photoinhibition, and modulating photosynthetic light harvesting. This mini-review presents an overview of recent important progress in site-directed mutagenesis studies of Cyt b 559 and implications for revealing the physiological functions of Cyt b 559 in PSII.

SUBMITTER: Chu HA

PROVIDER: S-EPMC4709441 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

The Roles of Cytochrome b 559 in Assembly and Photoprotection of Photosystem II Revealed by Site-Directed Mutagenesis Studies.

Chu Hsiu-An HA Chiu Yi-Fang YF

Frontiers in plant science 20160112

Cytochrome b 559 (Cyt b 559) is one of the essential components of the Photosystem II reaction center (PSII). Despite recent accomplishments in understanding the structure and function of PSII, the exact physiological function of Cyt b 559 remains unclear. Cyt b 559 is not involved in the primary electron transfer pathway in PSII but may participate in secondary electron transfer pathways that protect PSII against photoinhibition. Site-directed mutagenesis studies combined with spectroscopic and ...[more]

PMID: 26793230

Similar Datasets

Project description:BackgroundSite-directed mutagenesis (SDM) is a key method in molecular biology; allowing to modify DNA sequences at single base pair resolution. Although many SDM methods have been developed, methods that increase efficiency and versatility of this process remain highly desired.MethodWe present a versatile and simple method to efficiently introduce a variety of mutation schemes using Gibson-assembly but without the need to design uniquely designated Gibson primers. Instead, we explore the re-use of standard SDM primers (completely overlapping in sequence) in combination with regular primers (~ 25 bps long) for amplification of fragments flanking the site of mutagenesis. We further introduce a rapid amplification step of the Gibson-assembled product for analysis and quality control, as well as for ligation, or re-ligation at instances the process fails (avoiding expenditure of added Gibson reaction mixtures).ResultsWe first demonstrate that standard SDM primers can be used with the Gibson assembly method and, despite the need for extensive digestion of the DNA past the entire primer sequence, the reaction is attainable within as short as 15 min. We also find that the amount of the assembled Gibson product is too low to be visualized on standard agarose gel. Our added amplification step (by use of the same short primers initially employed) remedies this limitation and allows to resolve whether the desired Gibson-assembled product has been obtained on agarose gel or by sequencing of amplicons. It also provides large amounts of amplicons for subsequent ligations, bypassing the need to re-employ Gibson mixtures. Lastly, we find that our method can easily accommodate SDM primers with degenerate sequences.ConclusionWe employ our alternative approach to delete, replace, insert, and degenerate sequences within target DNA sequences, specifically DNA sequences that proved very resistant to mutagenesis by multiple other SDM methods (standard and commercial). Importantly, our approach involves the re-use of SDM primers from our primer-inventory. Our scheme thereby reduces the need (and time and money) to design and order new custom Gibson-primers. Together, we provide a simple and versatile protocol that spans only 4 days (including the added amplification step), requires minimal primer sets and provides very high yields and success rates (> 98%).

Project description: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 b 559 (Cyt b 559), 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 b 559, 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 Car D2 (∙+) ; Car D2 (∙+) 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.

Dataset Information

The Roles of Cytochrome b 559 in Assembly and Photoprotection of Photosystem II Revealed by Site-Directed Mutagenesis Studies.

Publications

The Roles of Cytochrome b 559 in Assembly and Photoprotection of Photosystem II Revealed by Site-Directed Mutagenesis Studies.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets