Assembly of D1/D2 complexes of photosystem II: Binding of pigments and a network of auxiliary proteins

Abstract

Photosystem II (PSII) is the multi-subunit light-driven oxidoreductase that drives photosynthetic electron transport using electrons extracted from water. To investigate the initial steps of PSII assembly, we used strains of the cyanobacterium Synechocystis sp. PCC 6803 arrested at early stages of PSII biogenesis and expressing affinity-tagged PSII subunits to isolate PSII reaction center assembly (RCII) complexes and their precursor D1 and D2 modules (D1mod and D2mod). RCII preparations isolated using either a His-tagged D2 or a FLAG-tagged PsbI subunit contained the previously described RCIIa and RCII* complexes that differ with respect to the presence of the Ycf39 assembly factor and high-light-inducible proteins (Hlips) and a larger complex consisting of RCIIa bound to monomeric PSI. All RCII complexes contained the PSII subunits D1, D2, PsbI, PsbE, and PsbF and the assembly factors rubredoxin A (RubA) and Ycf48, but we also detected PsbN, Slr1470, and the Slr0575 proteins, which all have plant homologs. The RCII preparations also contained prohibitins/stomatins (Phbs) of unknown function and FtsH protease subunits. RCII complexes were active in light-induced primary charge separation and bound chlorophylls, pheophytins, beta-carotenes, and heme. The isolated D1mod consisted of D1/PsbI/Ycf48 with some Ycf39 and Phb3, while D2mod contained D2/cytochrome b559 with co-purifying PsbY, Phb1, Phb3, FtsH2/FtsH3, CyanoP, and Slr1470. As stably bound chlorophyll was detected in D1mod but not D2mod, formation of RCII appears to be important for stable binding of most of the chlorophylls and both pheophytins. We suggest that chlorophyll can be delivered to RCII from either monomeric PSI or Ycf39/Hlip complexes.

FtsH Protease Inactivation Allows Accumulation of Aberrant Photosystem II in a Chlamydomonas Rubredoxin Mutant

“…Many amino acid residues in the DE loop of the D1 protein, which are also in the vicinity of the non-heme iron (F239, Q241, E242, Y246), have been previously reported to undergo oxidation (Kumar et al, 2021), and the amino acids between 238 and 248 have been proposed to be the region where cleavage of the DE loop occurs in vivo (Greenberg et al, 1987). A model in which RBD1 functions in iron delivery is consistent with the observation that the cyanobacterial RubA interacts with D1 during the formation of the D1-D2 heterodimeric complex RCII (Kiss et al, 2019) but is not present in either of the direct precursors of RCII, the so-called D1 mod and D2 mod subcomplexes (Knoppová et al, 2022). The formation of the RCII complex is presumably accompanied by the insertion of the non-heme iron, given that both D1 and D2 provide the ligands for coordinating this metal ( Fig.…”

Advances in the Understanding of the Lifecycle of Photosystem II

“…The PsbI subunit and cytochrome b559 (made up of the PsbE and PsbF subunits) complete the reaction center. This reaction center complex has been isolated independently and is the smallest PSII sub-complex capable of charge separation [ 19 , 20 , 21 ]. It is notable, however, that the CP43 subunit also provides a ligand to the manganese cluster, and so, while capable of charge separation, the reaction center proteins alone are not sufficient to bind the catalytic metal center or perform water-oxidizing chemistry.…”

“…The deletion of PratA was shown to be deficient in c-terminal processing of D1 [ 36 ], leading to the hypothesis that it facilitates access of the c-terminal protease, CtpA, to pD1. The rubredoxin RubA [ 21 , 31 , 38 ] also binds to D1 prior to its association with D2 and has been implicated in protection from photodamage to the reaction center during PSII assembly in the green alga Chlamydomonas reinhardtii [ 38 ].…”

“…Pam68 , along with Ycf48 and Ycf39 , is implicated in co-translational insertion of chlorophyll to the reaction center proteins D1 and D2 [ 28 , 37 , 43 , 44 ]. Ycf48 is a soluble thylakoid membrane-associated protein associated with the D1 pre-complex and the reaction center complex [ 21 , 27 , 31 , 45 , 46 , 47 , 48 , 49 ], and it contains a conserved arginine patch that mediates its binding to integral membrane proteins [ 48 ]. These findings, in addition to the finding that apo-D1 does not accumulate [ 29 ], provide evidence that chlorophyll is co-translationally inserted into D1 as it is translocated into the membrane.…”

“…PsbP (CyanoP), which binds to the D2 pre-complex [ 52 ], is thought to be involved in RC formation [ 22 , 53 , 54 ]. Recently, an RC complex was isolated from a strain of S6803 lacking CP47 that contains PsbN as well as the protein products of slr0575 and slr1470 [ 21 ]. This complex is on the de novo synthesis pathway, as active PSII does not form in this strain.…”