The de-epoxidase and epoxidase reactions of Mantoniella squamata (Prasinophyceae) exhibit different substrate-specific reaction kinetics compared to spinach

In vivo the prasinophyceaen alga Mantoniella squamata Manton et Parke uses an incomplete violaxanthin (Vx) cycle, leading to a strong accumulation of antheraxanthin (Ax) under conditions of high light. Here. we show that this zeaxanthin (Zx)-depleted Vx/Ax cycle is caused by an extremely slow second deepoxidation step from Ax to Zx, and a fast epoxidation from Ax back to V x in the light. The rate constant of Ax epoxidation is 5 to 6 times higher than the rate constant of Zx formation. implying that Ax is efficiently con verted back to Vx before it can be de-epoxidated to Zx. It is, however, onl y half the rate constant of the first deepoxidation step from Vx to Ax, thu s explaining the observed net accumulation of Ax during periods of strong i llumination. When comparing the rate constant of the second de-epoxidation step in M. squamata with Zx formation in spinach (Spinacia oleracea L.) thy lakoids, we find a 20-fold reduction in the reaction kinetics of the former . This extremely slow Ax de-epoxidation. which is also exhibited by the iso lated Mantoniella violaxanthin de-epoxidase (VIDE), is due to a reduced sub strate affinity of M. squamata VDE for Ax compared with the VDE of higher p lants. Mantoniella VDE, which has a similar K-m value for Vx, shows a subst antially increased K-m for the substrate Ax in comparison with spinach VDE. Our results furthermore explain why Zx formation in Mantoniella cells can only be found at low pH values that represent the pH optimum of VDE. A pH o f 5 blocks the epoxidation reaction and, consequently, leads to a slow but appreciable accumulation of Zx.