Nievelstein V, Vandekerchove J, Tadros M H, Lintig J V, Nitschke W, Beyer P
Biologisches Institut II, Zellbiologie, Freiburg, Germany.
Eur J Biochem. 1995 Nov 1;233(3):864-72. doi: 10.1111/j.1432-1033.1995.864_3.x.
The enzymic activity of phytoene desaturase in Narcissus pseudonarcissus chromoplast membranes depends in an essential way on the redox state of its environment. Here, the main redox-active components are quinones and tocopherols. Quinones (oxidized) act as intermediate electron acceptors in the desaturation reaction, as can be shown in reduced, hydroquinone-rich membranes. However, their complete oxidation by ferricyanide treatment of membranes leads to inhibition of the desaturation activity and, under these conditions, hydroquinones are required for reactivation. Using redox titrations, it is shown here that the optimal activity lies in the range of the midpoint potential of the plastoquinone/plastohydroquinone redox couple. For the adjustment of redox states of the redox-active lipid components in (photosynthetically inactive) chromoplasts, NADPH and oxygen are involved, the latter acting as a terminal acceptor. This results in a respiratory redox pathway in chromoplast membranes which is described here, to our knowledge, for the first time. Since phytoene desaturation responds to the redox state of quinones, which is adjusted by the respiratory redox pathway, the two reactions must be regarded as being mechanistically linked. The first protein component involved in the respiratory pathway which we have investigated molecularly is a 43-kDa NAD(P)H:quinone oxidoreductase, which is organized as a homodimer (23 +/- 3 kDa/subunit) and apparently possesses a manganese redox center. Internal protein microsequencing and cloning of the corresponding cDNA revealed a high degree of similarity to the 23-kDa protein of the oxygen-evolving complex of photosystem II, but no information about the N-terminal organization of the oxidoreductase could be obtained. During flower development, the steady-state concentration of the corresponding mRNA is up-regulated, indicating a specific function of the gene product in chlorophyll-free chromoplasts.
水仙质体色素体膜中八氢番茄红素去饱和酶的酶活性在本质上取决于其所处环境的氧化还原状态。在此，主要的氧化还原活性成分是醌类和生育酚。醌类（氧化态）在去饱和反应中作为中间电子受体，这在还原态、富含对苯二酚的膜中可以得到证明。然而，用铁氰化物处理膜使其完全氧化会导致去饱和活性受到抑制，在这些条件下，需要对苯二酚来重新激活。通过氧化还原滴定法表明，最佳活性处于质体醌/质体对苯二酚氧化还原对的中点电位范围内。对于（光合不活跃的）质体色素体中氧化还原活性脂质成分氧化还原状态 的调节，涉及到NADPH和氧气，后者作为终端受体。据我们所知，这在此处首次描述了质体色素体膜中的一种呼吸氧化还原途径。由于八氢番茄红素去饱和反应对醌类的氧化还原状态有响应，而醌类的氧化还原状态是由呼吸氧化还原途径调节的，所以这两个反应在机制上必定是相互关联的。我们在分子水平上研究的参与呼吸途径的第一个蛋白质成分是一种43 kDa的NAD(P)H：醌氧化还原酶，它以同二聚体（23±3 kDa/亚基）形式存在，并且显然具有一个锰氧化还原中心。内部蛋白质微测序和相应cDNA的克隆显示，它与光系统II放氧复合体的23 kDa蛋白质有高度相似性，但关于氧化还原酶N端结构的信息无法获得。在花发育过程中，相应mRNA的稳态浓度上调，表明该基因产物在无叶绿素的质体色素体中有特定功能。
