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首页> 外文期刊>The New Phytologist >Functional characterization of the chloroplast ferric chelateoxidoreductase enzyme
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Functional characterization of the chloroplast ferric chelateoxidoreductase enzyme

机译：叶绿体铁螯合氧化还原酶的功能表征

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摘要

Iron (Fe) has an essential role in the biosynthesis of chlorophylls and redox cofactors, andthus chloroplast iron uptake is a process of special importance. The chloroplast ferric chelateoxidoreductase (cFRO) has a crucial role in this process but it is poorly characterized. To study the localization and mechanism of action of cFRO, sugar beet (Beta vulgaris cvOrbis) chloroplast envelope fractions were isolated by gradient ultracentrifugation, and theirpurity was tested by western blotting against different marker proteins. The ferric chelatereductase (FCR) activity of envelope fractions was studied in the presence of NAD(P)H(reductants) and FAD coenzymes. Reduction of Fe(III)-ethylenediaminetetraacetic acid wasmonitored spectrophotometrically by the Fe(II)-bathophenanthroline disulfonate complexformation. FCR activity, that is production of free Fe(II) for Fe uptake, showed biphasic saturationkinetics, and was clearly associated only to chloroplast inner envelope (cIE) vesicles. The reactionrate was > 2.5 times higher with NADPH than with NADH, which indicates the naturalcoenzyme preference of cFRO activity and its dependence on photosynthesis. FCR activity of cIE vesicles isolated from Fe-deficient plants also showed clear biphasickinetics, where the KM of the low affinity component was elevated, and thus this componentwas down-regulated.
机译：铁（Fe）在叶绿素和氧化还原辅助因子的生物合成中起着至关重要的作用，因此叶绿体铁的吸收是一个特别重要的过程。叶绿体三价铁螯合氧化还原酶（cFRO）在此过程中起着至关重要的作用，但其表征较差。为了研究cFRO的定位及其作用机理，通过梯度超速离心分离了甜菜（Beta vulgaris cvOrbis）叶绿体包膜部分，并通过蛋白质印迹法针对不同的标记蛋白测试了其纯度。在存在NAD（P）H（还原剂）和FAD辅酶的情况下研究了包膜部分的铁螯合还原酶（FCR）活性。 Fe（II）-二苯并菲咯啉二磺酸盐配合物分光光度法监测Fe（III）-乙二胺四乙酸的还原。 FCR活性，即为摄取铁而产生的游离Fe（II），显示出两相饱和动力学，并且显然仅与叶绿体内膜（cIE）囊泡相关。使用NADPH的反应速率比使用NADH的反应速率高> 2.5倍，这表明cFRO活性对天然酶的偏爱及其对光合作用的依赖性。从缺铁植物中分离的cIE囊泡的FCR活性也显示出明显的双相运动，其中低亲和力组分的KM升高，因此该组分被下调。

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