Sequence analysis of photoaffinity‐labelled peptides derived by proteolysis of photosystem‐2 reaction centres from thylakoid membranes treated with [¹⁴C]azidoatrazine

Abstract

Photosystem-2 reaction centres were prepared from pea thylakoid membranes that had been photoaffinity labelled with [14C]-azidoatrazine (2-azido-4-ethylamino-6-isopropylamino-s-triazine), a derivative of the herbicide atrazine which binds to the secondary plastoquinone electron-acceptor site of photosystem 2. SDS/PAGE of the 14C-labelled reaction centres followed by fluorography revealed photoaffinity-labelled proteins of apparent molecular masses 30 kDa and 55 kDa, which corresponded to the D1 polypeptide and to an SDS-stable heterodimer of the D1 and D2 polypeptides, respectively. To obtain sequence information on the site of photoaffinity labelling, an 8-kDa photoaffinity-labelled peptide, generated by proteolysis of the reaction-centre material with trypsin, was isolated and purified to apparent homogeneity using reverse-phase and size-exclusion HPLC techniques. The amino terminus of the photoaffinity-labelled peptide was determined to be LeuGly-Met-Arg-Pro-Xaa-Ile-Ala-Val-Ala-Tyr by Edman sequencing. This corresponds to the amino terminus of a predicted tryptic peptide of D1 and confirms that azidoatrazine photolabels the D1 polypeptide of photosystem 2 in the region Leu1 37 -Arg225. Chymotrypsin/trypsin digestion of photoaffinity-labelled reaction centres followed by reverse-phase HPLC was used to isolate a smaller photoaffinity-labelled peptide. On Edman sequencing, Ser-Ala were identified as the first two residues and 14C was released on the third cycle, after which further degradation was blocked. The two potential peptide fragments with Ser-Ala at the amino terminus in the region Leu137 -Arg225 are Serl48-Ala-Pro and Ser212-Ala-Met. Proline is an unlikely target for reaction with the nitrene of the photoactivated azidoatrazine, and the data are thus consistent with Met214 as the site of photoaffinity labelling on D1 when thylakoid membranes are illuminated with ultraviolet irradiation in the presence of [14C]azidoatrazine.Most of the herbicides which block electron transport through photosystem 2 (PS2) bind in competition with plastoquinone at the so-called secondary acceptor plastoquinone (QB) site on PS2, thus blocking the oxidation of the reduced primary acceptor plastoquinone QA by the secondary acceptor plastoquinone Qe [l -31. The specific interactions of these herbicides with amino acid residues around the QB site are of interest because information on these interactions may assist the design of novel inhibitors with herbicidal activity and it may also aid our understanding of the reactions of plastoquinone at the QB site in vivo. The herbicide receptor protein in PS2 was identified as the D1 polypeptide using azidoatrazine, a photoaffinity-labelling analogue of the herbiCorrespondence to J. R. Bowyer, Department of Biochemistry, Royal Holloway and Bedford New College, University of London, Egham Hill, Egham, Surrey, TW20 OEX, EnglandAbbreviations. Atrazine, 2-chloro-4-ethylamino-6-isopropylamino-s-triazine; azidoatrazine, 2-azido-4-ethylamino-6-isopropylamino-s-triazine; BBY, photosystem-2-enri...

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