Isolation and characterization of photosystem II subcomplexes from cyanobacteria lacking photosystem I

Abstract

A photosystem II (PSII) core complex lacking the internal antenna CP43 protein was isolated from the photosystem II of Synechocystis PCC6803, which lacks photosystem I (PSI). CP47-RC and reaction centre (RCII) complexes were also obtained in a single procedure by direct solubilization of whole thylakoid membranes. The CP47-RC subcore complex was characterized by SDS/PAGE, immunoblotting, MALDI MS, visible and fluorescence spectroscopy, and absorption detected magnetic resonance. The purity and functionality of RCII was also assayed. These preparations may be useful for mutational analysis of PSII RC and CP47 -RC in studying primary reactions of oxygenic photosynthesis.Keywords: CP47 -RC subcomplex; cyanobacteria; photosystem II.The photosystem II (PSII) of oxygenic photosynthetic organisms is composed of more than 25 protein subunits and binds a large number of pigments. Primary charge separation occurs at the reaction centre (RCII) consisting of D1, D2, a and b subunits of cytochrome b559, PsbI and PsbW [1 -3]. CP47 and CP43, the two internal antenna chlorophyll a-binding proteins are closely associated with the D1/D2 heterodimer. The main light-harvesting system is formed of chlorophyll a/b binding proteins in higher plants and green algae, and of phycobilisomes in red algae and cyanobacteria. A 'core complex' consisting of D1, D2, CP47, CP43 and other subunits may be obtained by removing the main light harvesting system from PSII. Treatment of the PSII core complex with detergents can 'peel' away various subunits. Recent structural studies employing electron micrographic techniques have revealed that the CP43 protein is located closer to the surface of the complex than CP47 [4]. Indeed, CP43 is more easily removed during detergent treatment, at least in higher plants, allowing isolation of the CP47-RC complex. RCII and CP47 -RC from higher plants have been prepared according to several different procedures [1,[5][6][7][8].The isolation and characterization of these PSII core subcomplexes from wild-type and mutant higher plants represents an important step towards our understanding of the structure -function relationship of PSII. However, sitedirected mutagenesis of PSII in plants is hampered by the difficulty of chloroplast transformation. The unicellular cyanobacterium Synechocystis PCC6803 is generally used as a model organism for the study of PSII. Of importance is the fact that this species is able to grow photoheterotrophically and is easily transformable, allowing the construction of mutants defective in PSII function [9]. However, their thylakoid membrane contains five times more PSI than PSII, hindering both biophysical analysis and the purification of PSII. This problem has been resolved by constructing a mutant in which part of the psaAB operon coding for the core proteins of PSI is deleted [10]. PSI-less cells have approximately six times more PSII than wild-type cells, as estimated on a chlorophyll basis [10].Attempts to isolate CP43-less PSII core complexes [11] as well as RCII from cyanobacteria...

Accumulation of the D2 Protein Is a Key Regulatory Step for Assembly of the Photosystem II Reaction Center Complex in Synechocystis PCC 6803

“…The complex has been found in similar mutants (54,55) and a significantly elevated psbA transcript level had been noted (24). Our finding that the highest label in D1 is observed in the RC47 complex while only a minimal amount of the D1 protein was found in RC complexes as well as in the free fraction is remarkable.…”