Isolation of the photosystem II reaction center complex from barley. Characterization by circular dichroism spectroscopy and amino acid sequencing

Abstract

A photosystem II (PSII) reaction center complex containing Chla -protein 2, Chla -protein 3, cytochrome b559, the herbicide-binding polypeptide and the 33 kD polypeptide from the oxygen-evolving site of PSII was isolated by sucrose gradient centrifugation of the dodecyl-β, d-maltoside extract of grana membranes highly enriched in PSII. The reaction center complex contained ca 19 lipid molecules per reaction center and sedimented slightly faster than catalase. It mediated light-induced, DCMU-sensitive electron transport from 1,5-diphenylcarbazide to dichlorophenolindophenol. The N-terminal amino acid sequence of a proteolytic fragment of Chla -protein 2 was determined. Eighteen out of 20 amino acid residues were identical with the deduced amino acid sequence of the 51 kD Chla -apoprotein from spinach. In contrast to grana membranes, which had a 77 K fluorescence emission maximum at 685 nm with a pronounced shoulder at 695 nm, the PSII reaction center complex had a single peak at 685 nm. Circular dichroism measurements in the visible range showed that the pigments associated with the isolated PSII reaction center complex and the light-harvesting complex (LHC) were highly oriented. The secondary structure of the PSII reaction center complex and LHC was investigated by circular dichroism measurements in the ultraviolet range. Both had a high α-helix content, suggesting that the chlorophyll molecules may be oriented between transmembrane helices in a similar way as in bacterial reaction centers and light harvesting systems. © 1985 Carlsberg Laboratory.