EPR characterization of an oxygen-evolving photosystem II preparation from the transformable cyanobacterium Synechocystis 6803

Abstract

The transformable cyanobacterium Synechocystis 6803 has a photosynthetic apparatus that is similar to that of plants. Because of the ease with which this organism can be genetically manipulated and isotopically labeled, Synechocystis has been used extensively in recent studies of electron transfer in the water-splitting complex, photosystem II. Here, we present the first EPR characterization of a highly active oxygen-evolving preparation from this organism. This preparation shows oxygen-evolution activities in the range from 2400-2600 mumol of O2/(mg of chlorophyll.h). We show that this preparation is stable enough for room temperature EPR studies. We then use this assay to show that the lineshapes of the D+ and Z+ tyrosine radicals are identical in this preparation, as has been observed in photosystem II complexes from a wide variety of photosynthetic species. We also present the first multiline EPR spectrum that has been observed from the Synechocystis manganese cluster.

Mutations in the CP43 Protein of Photosystem II Affect PSII Function and Cytochrome C550 Binding

“…The minor peaks arise from accessory pigment molecules associated with the photosystem. These spectra are similar to spectra obtained from PSII preparations of non-His-tagged control strains (Noren et al, 1991;Tang & Diner, 1994) and to spectra obtained from other His-tagged PSII preparations (Bricker et al, 1998).…”

Amine binding and oxidation at the catalytic site for photosynthetic water oxidation

“…Cl Ϫ depletion of PSII-1, PSII-2, and PSII-3 was performed by repeated homogenization into buffer A (remaining Cl Ϫ Ͻ1.6 mM). Spinach MSP (manganese stabilizing protein) (24), spinach LHC (lightharvesting complex) (23), and cyanobacterial PSI (photosystem I) and PSII (25) samples were isolated by procedures previously described. These samples were exchanged into buffer A, and Cl Ϫ was depleted by several rounds of concentration with a Centricon 10 or 100 (Amicon) and subsequent dilution with buffer A. O 2 evolution and chlorophyll assays were performed by methods described previously (26).…”

Synechocystis sp PCC 6803 strains lacking photosystem I and phycobilisome function.

“…However, detailed functional and biochemical characterization of PSll mutants generally is complex because the PSlllPSl reaction center ratio is unfavorable in this cyanobacterium (Fujita and Murakami, 1988). Even though severa1 useful PSll preparation procedures are available for wild-type Synechocystis 6803 (Burnap et al, 1989;Noren et al, 1991;Kirilovsky et al, 1992;Nilsson et al, 1992), preparation of oxygen-evolving PSll particles from a number of mutants has been unsuccessful, possibly due to a destabilized oxygenevolving complex in these mutants. Apart from PSI, the presente of phycobilisome components may also complicate the To whom correspondence should be addressed.…”