Site-directed Mutations at Residue 251 of the Photosystem II D1 Protein of Chlamydomonas That Result in a Nonphotosynthetic Phenotype and Impair D1 Synthesis and Accumulation

Abstract

In Cyanobacteria and Chlamydomonas reinhardtii, substitution of valine for alanine at position 251 of the photosystem II D1 protein in the loop between transmembrane helices IV and V confers resistance to herbicides that reduce photosystem II function and increases sensitivity to photoinhibition. Using site-directed mutagenesis and chloroplast transformation in Chlamydomonas we have examined further the role of residue 251 in relation to D1 structure, function, and photosynthetic performance. Of the 12 different amino acid substitutions for Ala 251 introduced at this position, five (Arg, Asp, Gln, Glu, and His) resulted in a nonphotosynthetic phenotype. Transformants with the Arg 251 substitution synthesize a normal sized 32-kDa D1 protein with greatly reduced stability. The Gln, Glu, His, and Asp transformants make a 33-34-kDa form of the D1 protein of varying stability as well as an immunologically related polypeptide of 24 -25 kDa corresponding to the N-terminal portion of D1 that is unstable and appears to be an aborted D1 translation product. All mutant forms of the D1 protein are intrinsic to the thylakoids. In contrast to previous studies in Cyanobacteria showing that residues in the IV-V loop can be mutated or deleted without loss of photosynthetic competence, our results suggest that Ala 251 has a key role in the structure and function of the IV-V loop region.In all oxygen-evolving organisms, the reaction center of the photosystem II (PSII) 1 complex consists of the D1 protein, the structurally related D2 protein, cytochrome b 559 , and at least two small proteins of unknown function (1). The rapidly turning over D1 protein continuously undergoes a cycle of damage, degradation, and replacement in response to photodamage from normal PSII photochemistry (2). The D1 protein, encoded by the chloroplast psbA gene, is synthesized as a membrane associated 33.5-kDa precursor with a C-terminal extension. After processing to the 32-kDa mature form, D1 undergoes several posttranslational modifications postulated to facilitate translocation and proper assembly into PSII centers in the lipid bilayer (3), where it binds chlorophyll, pheophytin, quinone, carotenoid, iron, and manganese (4). D1 is thought to have five hydrophobic membrane-spanning helices, with its N terminus facing the chloroplast stroma and its C terminus projecting into the thylakoid lumen (5, 6). One stromally exposed region of D1, extending from the C terminus of helix IV through the N terminus of helix V (IV-V loop), participates in binding both Q B , the second stable quinone acceptor in PSII, and several classes of herbicides that inhibit photosynthetic electron transport (6). The IV-V loop region includes a stromal helix thought to lie parallel to the membrane surface (6) that divides this loop into two parts, one thought to be involved in D1 degradation in vivo and the other functioning in herbicide and quinone binding (7,8).Phylogenetic conservation of the IV-V loop among Cyanobacteria, algae, and higher plants (9) suggests that most of thes...

Biophysical, Biochemical, and Physiological Characterization ofChlamydomonas reinhardtii Mutants with Amino Acid Substitutions at the Ala251 Residue in the D1 Protein That Result in Varying Levels of Photosynthetic Competence

“…This loop, whose amino acid sequence is highly conserved among cyanobacteria, algae, and higher plants (7), is involved in binding both Q B , the second stable quinone acceptor in PSII, and several classes of herbicides that inhibit photosynthetic electron transport at the Q B docking site (8). Only a few of the many amino acid substitutions made in this region result in the loss of D1 function and photosynthetic capability (9,10), suggesting that most positions can tolerate considerable variation in R group conformation or charge and still permit D1 function. However, particular residues may be necessary for optimal PSII activity or may provide functional advantages under certain environmental conditions (11,12).…”

“…Five of these substitutions (Arg, Asp, Gln, Glu, and His) were shown to lead to a nonphotosynthetic phenotype and to impaired D1 synthesis and accumulation (10). Seven (Cys, Gly, Ile, Leu, Pro, Ser, and Val), which retain photosynthetic function to various degrees, affect herbicide resistance and photoautotrophic growth rates to various degrees (17).…”

“…Each amino acid alteration at position 251 is linked to a silent SspI RFLP marker (*) in the codon of residue 247 and all genotypes contained the selectable spectinomycin resistance marker in the 16 S rRNA (10).…”

“…In Vitro Site-directed Mutagenesis and Chloroplast Transformation of C. reinhardtii-The A251A* (wild type control, CC-3394, GCT), and D1 mutants A251G* (CC-3393, GGT), A251I* (CC-3388, ATT), A251V* (CC-3387, GTT), A251R* (CC-3376, CGT), A251Q* (CC-3374, CAA), A251E* (CC-3377, GAA), A251H* (CC-3378, CAC), and A251D* (CC-3375, GAC) were obtained by site-directed mutagenesis of exon 4 of the chloroplast psbA gene followed by cotransformation as described previously (10,17). Each amino acid alteration at position 251 is linked to a silent SspI RFLP marker (*) in the codon of residue 247 and all genotypes contained the selectable spectinomycin resistance marker in the 16 S rRNA (10).…”

Tansley Review No. 112

“…These enzymes reduce organic peroxides to their corresponding alcohols and might be crucial in controlling chain-type reactions that follow the initiation of lipid peroxidation by singlet O # or hydroxyl radical. Intensive study has been devoted to modification of D1 by the use of mutagenetic techniques (Gleiter et al, 1992 ;Perewoska et al, 1994 ;Nixon et al, 1995 ;Minagawa et al, 1996 ;Lardans et al, 1997 ;Ma$ enpa$ a$ et al, 1998 ;Mulo et al, 1998). None of these has elucidated the role of AOS at specific sites within PSII.…”