and functions in de novo assembly and repair of

Photosystem II (PSII) undergoes frequent photooxidative damage that, if not repaired, impairs photosynthetic activity and growth. How photosynthetic organisms protect vulnerable PSII intermediate complexes during de novo assembly and repair remains poorly understood. Here, we report the genetic and biochemical charac- terization of chloroplast-located rubredoxin 1 (RBD1), a PSII assembly factor containing a redox-active rubredoxin domain and a single C-terminal transmembrane α -helix (TMH) domain. RBD1 is an inte-gral thylakoid membrane protein that is enriched in stroma lamellae fractions with the rubredoxin domain exposed on the stromal side. RBD1 also interacts with PSII intermediate complexes containing cytochrome b 559 . Complementation of the Chlamydomonas reinhardtii (hereafter Chlamydomonas ) RBD1-deficient 2pac mutant with constructs encoding RBD1 protein truncations and site-directed muta- tions demonstrated that the TMH domain is essential for de novo PSII assembly, whereas the rubredoxin domain is involved in PSII repair. The rubredoxin domain exhibits a redox midpoint potential of + 114 mV and is proficient in 1-electron transfers to a surrogate cytochrome c in vitro. Reduction of oxidized RBD1 is NADPH dependent and can be mediated by ferredoxin-NADP + reductase (FNR) in vitro. We propose that RBD1 participates, together with the cytochrome b 559 , in the protection of PSII intermediate complexes from photooxidative damage during de novo assembly and repair. This role of RBD1 is consistent with its evolutionary conservation among photosynthetic organisms and the fact that it is essential in photosynthetic eukaryotes. 5 Plant Growth and Chloroplast Isolation. Arabidopsis Columbia ecotype (Col-0) seedlings (WT) were grown on soil under 100 μ mol photons · m − 2 · s − 1 with 10-h light/14-h dark conditions at 22 °C for 5 to 6 wk. Spinach leaves were obtained from a local market. For intact chloroplast isolation, plants were homogenized with an ULTRA-TURRAX disperser homogenizer (IKA) in chloroplast isolation buffer (20 mM Hepes-KOH, pH 8, 330 mM sorbitol, 10 mM EDTA, 10 mM NaHCO 3 , and 5 mM MgCl 2 ). The homogenate was filtered through a double layer of Miracloth (Calbiochem) and collected in a prechilled Erlenmeyer flask. The obtained filtrate was centrifuged at 1,300 × g for 8 min at 4 °C to pellet intact chloroplasts. Pelleted chloroplasts were resuspended in resuspension buffer (20 mM Hepes-KOH, pH 8, 330 mM sorbitol, 10 mM NaHCO 3 , and 5 mM MgCl 2 ) and loaded onto a premade, 40% and 75%, 2-step Percoll (GE Healthcare) gradient. Chloroplasts loaded onto the Percoll gradient were centrifuged at 1,500 × g in a swing-out rotor for 10 min at 4 °C, and chloroplasts were collected from the 40 to 75% green band intersection. Collected chloroplasts were diluted with 3 vol of resuspension buffer and further pelleted by centrifugation at 1,200 × g for 5 min at 4 °C. Chloroplast densities and intactness were determined with a Multisizer3 (Beckman Coulter). TMs from intact chloroplasts were isolated by lysing with hypoosmotic lysis buffer (20 mM Hepes/KOH, pH 8.0, 5 mM MgCl 2 ) and collected by centrifugation