Photosystem II Peripheral Accessory Chlorophyll Mutants inChlamydomonas reinhardtii. Biochemical Characterization and Sensitivity to Photo-Inhibition,

Abstract

In addition to the four chlorophylls (Chls) involved in primary charge separation, the photosystem II (PSII) reaction center polypeptides, D1 and D2, coordinate a pair of symmetry-related, peripheral accessory Chls. These Chls are axially coordinated by the D1-H118 and D2-H117 residues and are in close association with the proximal Chl antennae proteins, CP43 and CP47. To gain insight into the function(s) of each of the peripheral Chls, we generated site-specific mutations of the amino acid residues that coordinate these Chls and characterized their energy and electron transfer properties. Our results demonstrate that D1-H118 and D2-H117 mutants differ with respect to: (a) their relative numbers of functional PSII complexes, (b) their relative ability to stabilize charge-separated states, (c) light-harvesting efficiency, and (d) their sensitivity to photo-inhibition. The D2-H117N and D2-H117Q mutants had reduced levels of functional PSII complexes and oxygen evolution capacity as well as reduced light-harvesting efficiencies relative to wild-type cells. In contrast, the D1-H118Q mutant was capable of near wild-type rates of oxygen evolution at saturating light intensities. The D1-H118Q mutant also was substantially more resistant to photo-inhibition than wild type. This reduced sensitivity to photo-inhibition is presumably associated with a reduced light-harvesting efficiency in this mutant. Finally, it is noted that the PSII peripheral accessory Chls have similarities to a to a pair of Chls also present in the PSI reaction center complex.Photosystem II (PSII) is a membrane-bound pigment-protein complex that catalyzes the light-driven oxidation of water and reduction of plastoquinone. The simplest functional PSII complex capable of charge separation is the reaction center complex. The PSII reaction center complex contains five polypeptides (Nanba and Satoh, 1987;Gounaris et al., 1990). The largest polypeptides (32 kD) are the D1 and D2 polypeptides, each of which has five transmembranespanning alpha helices. Sandwiched between the D1 and D2 polypeptides are the four chlorophylls (Chls) and two pheophytins (Pheos) involved in primary charge separation (for review, see Ruffle and Sayre, 1998). Three additional small (10 kD) polypeptides are present in the PSII reaction center complex. Two of these proteins, psbE and psbF, coordinate the redox active heme, cytochrome (Cyt) b 559 . This heme is not involved in primary charge separation but participates in a low quantum yield electron transfer cycle around PSII that protects the complex from photodamage (Buser et al., 1992; Stewart et al., 1998). The fifth protein component is the psbI protein, a small structural polypeptide that stabilizes the complex (Nanba and Satoh, 1987).Amino acid sequence similarities between the D1 and D2 proteins and the analogous L and M subunits of the bacterial (Rhodopseudomonas viridis) photosynthetic reaction center indicated that the PSII reaction center was structurally analogous to the bacterial photosynthetic reaction center (Deisen...

Functional asymmetry of photosystem II D1 and D2 peripheral chlorophyll mutants of Chlamydomonas reinhardtii

“…Although it was not possible to make direct comparisons between equivalent amino acid substitutions in D1 and D2 mutant RC particles (D2-H117Q RCs were not stable), it was evident that the energetic coupling between P680 and the peripheral Chl in the D1-H118Q mutant was more similar to WT, consistent with results obtained from Chl fluorescence steady-state and lifetime measurements (ref. 41; Figs. 1-3).…”

“…A change in orientation of the Chl, however, may alter potential protein-Chl interactions with other nearby residues (see CD spectra discussion below), accounting for the line-width broadening. In addition to their role in electron transfer processes, the peripheral accessory Chls have been shown to participate in energy transfer between the proximal antennae Chl protein complexes and P680 (14,19,20,41). To characterize the energetic couplings in PSII RCs, we measured the Chl (visible) CD spectra of WT and mutant RCs.…”

“…The modified psbA gene was delivered into a psbA-deficient C. reinhardtii strain, CC-741, as described by Minagawa and Crofts (42). D2-H117N and D2-H117Q mutants were generated as described (41). All of the mutations were confirmed by sequencing DNA from transgenic algae.…”

“…Thylakoids were prepared as described (41). PSII membrane fragments were prepared from C. reinhardtii similar to the methods of Berthold et al (43).…”

“…Additional evidence also suggests that the two peripheral accessory Chls are functionally different (41). Biochemical analyses indicate that D1-H118Q mutants have nearly wild-type (WT) rates of oxygen evolution at saturating light intensities whereas D2-H117Q mutants are light-saturated at relatively low light intensities.…”