The modification of atrazine binding by the redox state of the endogenous high-spin iron and by specific proteolytic enzymes in Photosystem II membrane fragments and intact thylakoids

Atrazine-binding properties reflecting the fine structure of the binding niche within the 32 kDa polypeptide, D1, were analyzed in thylakoids and Photosystem II membrane fragments from spinach as a function of two parameters: (i) the redox state of the endogenous iron located between Q_A and Q_B at the Photosystem II acceptor side, and (ii) proteolytic degradation by the specifically acting enzymes trypsin, glutaminic acid-specific, lysine-specific and arginine-specific proteinases. It was found (a) Fe²⁺-oxidation by K₃[Fe(CN)₆] significantly reduces atrazine binding in Photosystem II membrane fragments. The K₃[Fe(CN)₆] effect exhibits a marked pH dependence attributable to different percentages of Fe³⁺ formation. Thylakoids hardly show any modification of atrazine binding by K₃[Fe(CN)₆]; (b) proteolytic enzymes which are expected to interact with specific sites of the stroma-exposed loop between transmembrane helices IV and V of polypeptide D1 affect the atrzine-binding properties quite differently. The protective action of CaCl₂ to proteolytic degradation markedly depends on the nature of the enzyme used; (c) the degradation of the atrazine binding by a lysine-specific proteinase in samples from spinach, containing a lysine-free D1 polypeptide, is largely retarded if the atrazine-binding sites are occupied during the proteolytic treatment. The mechanistic implications of these findings are discussed.