The D1 C-terminal Processing Protease of Photosystem II fromScenedesmus obliquus

Abstract

Polypeptide D1 of the photosystem II reaction center of oxygenic photosynthesis is expressed in precursor form (pre-D1), and it must be proteolytically processed at its C terminus to enable assembly of the manganese cluster responsible for photosynthetic water oxidation. A rapid and highly sensitive enzyme-linked immunosorbent assay-based microtiter plate method is described for assaying this D1 C-terminal processing protease. A protocol is described for the isolation and purification to homogeneity of the enzyme from the green alga, Scenedesmus obliquus. Amino acid sequence information on the purified protease was used to clone the corresponding gene, the translated sequence of which is presented. A comparison of the gene product with homologous proteases points to a region of conserved residues that likely corresponds to the active site of a new class of serine protease. The LF-1 mutant strain of Scenedesmus (isolated by Dr. Norman Bishop) is incapable of processing pre-D1. We show here that the C-terminal processing protease gene in this strain contains a single base deletion that causes a frame shift and a premature stop of translation within the likely active site of the enzyme. A suppressor strain, LF-1-RVT-1, which is photoautotrophic and capable of processing pre-D1 has a nearby single base insertion that restores the expression of active enzyme. These observations provide the first definitive proof that the enzyme isolated is responsible for in vivo proteolytic processing of pre-D1 and that no other protease can compensate for its loss.The photosystem II reaction center contains two homologous polypeptides, D1 and D2, that are responsible for the coordination of the primary photoreactants (1, 2). D1 has also been shown to harbor the redox-active tyrosine, Tyr Z (3, 4), that links the oxidized primary electron donor of photosystem II to the oxygen-evolving manganese cluster and is thought to provide at least some of the coordinating ligands to this complex (5-8). D1 is expressed in precursor form (9 -11), inserted into the thylakoid membrane, and processed at its C terminus (6, 12-15) by a proteolytic enzyme, D1 C-terminal processing protease (Ctp-protease).1 In cyanobacteria, 16 residues are cleaved from precursor D1 (6), 9 in higher plants (15, 16), and 8 in Chlamydomonas 2 with processing occurring in all cases at the carboxyl side of D1-Ala-344. Euglena is the sole oxygenic photosynthetic organism that is believed not to undergo C-terminal processing (1), although, in this case, no C-terminal extension is present. Based on a comparison of 43 psbA genes, the amino acid sequence on the amino side of the processing site is strictly conserved within the 8 residues immediately upstream of the processing site (1). On the carboxyl side of the processing site, no residue is conserved throughout all 43 sequences. While position 345 is normally occupied by either an alanine or a serine, replacement of Ser-345 in Synechocystis 6803 with arginine or alanine (6) or in Chlamydomonas with glycine, cysteine, valine,...

The initial steps of biogenesis of cyanobacterial photosystems occur in plasma membranes

“…As mentioned earlier, the CtpA enzyme is absolutely required for the assembly of the wateroxidizing machinery in PSII. Mutants of Synechocystis 6803 (20), as well as the eukaryotic alga Scenedesmus obliquus (35,36), without functional CtpA cannot process the carboxyl-terminal extension of the pD1 protein. In these mutants, the PSII complex is assembled, and electron transfer from Y Z to Q B (Fig.…”

Photoassembly of the water-oxidizing complex in photosystem II

“…The D1 C-terminal extension was shown not to be required for assembly of functional PS II complexes, nor for growth under optimal conditions in both Synechocystis 6803 [37] and Chlamydomonas [38]. However, the carboxyl extension must be cleaved beyond Ala 344 by a soluble protease, called ctpA, prior to assembly of an active inorganic core [37,39]. This cleavage is essential for the assembly of the Mn 4 Ca-core and, hence, oxygen evolution.…”

Evidence that D1 Processing Is Required for Manganese Binding and Extrinsic Protein Assembly into Photosystem II

“…Studies of D1 processing mutants have revealed that processing of pD1 is essential for the assembly of a functional manganese cluster. However, the exact number of manganese atoms associated with pD1-containing PSII complexes has been the subject of debate (12)(13)(14)(15). Manganese measurements of membranes of the Scenedesmus obliquus LF1 mutant suggests that pD1-containing PSII complexes have only 1-2 manganese/PSII (16).…”