The Influence of N,N’-Dicyclohexylcarbodiimide (DCCD) on the Kinetics and pH-Dependence of Zeaxanthin Formation in Isolated Pea (Pisum sativum) Thylakoids

Abstract

The de-ep oxidation of violaxanthin (Viol) to antheraxanthin (Anth) and zeaxanthin (Zeax) in the xanthophyll cycle of higher plants [1] and the generation of pH-dependent fluorescence quenching (qE) in the antenna of photosystem H [2] are regulated by the pH of the thylakoid lumen. The physiological relevance of the xanthophyll de-epoxidation is based on a possible fimction of Zeax in thermal dissipation of excess light energy within photosystem H (PSH). It has been supposed that the decline of the lumenal pH in the light is accompanied by conformational changes in PSH antenna proteins which in turn induce thermal dissipation of absorbed light (qE-quenching of chlorophyll (CM) a fluorescence) [2]. Zeax may be involved in this photoprotective mechanism either by direct quenching of energy [3] or by supporting conformational changes of antenna proteins [2]. The Vio de-epoxidase (VDE) has been identified as a nuclear-encoded 43 kDa protein which is located in the thylakoid lumen [4,5]. The enzyme requires ascorbate as a cofactor and shows an optimum pH of 5.2 [6]. The pH-dependent activation of VDE is accompanied by binding of the enzyme to the membrane (see [7] for a review). Incubation of isolated thylakoids with N,N’-Dicyclohexylcarbodiimide (DCCD) inhibits the generation of qE [8]. DCCD is known to bind to nearly all antenna proteins of both photosystems [9,10]. It has been suggested that binding of DCCD to lumen exposed carboxy groups of PSH antenna proteins blocks protonation and therefore energy dissipation in PSH [2]. In this work, we investigated the influence of DCCD on the de-epoxidation reactions of the xanthophyll cycle in isolated pea thylakoids. To allow discrimination between binding of DCCD to antenna proteins and other proteins we studied in parallel the reactions in thylakoids from intermittent light (IML) grown plants which lack most of the antenna proteins [11].