Measurement of ferrochelatase activity using a novel assay suggests that plastids are the major site of haem biosynthesis in both photosynthetic and non-photosynthetic cells of pea (Pisum sativum L.)
Measurement of ferrochelatase activity using a novel assay suggests that plastids are the major site of haem biosynthesis in both photosynthetic and non-photosynthetic cells of pea (Pisum sativum L.)
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DOI：
10.1042/0264-6021:3620423
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发表时间：
2002-03-01
影响因子：
4.1
通讯作者：
Smith, AG
中科院分区：
文献类型：
--
作者：
Cornah, JE;Roper, JM;Smith, AG
亚铁螯合酶是血红素生物合成的末端酶，催化亚铁插入原卟啉IX的大环中，原卟啉IX是血红素和叶绿素合成的最后一个共同中间体。在高等植物的质体和线粒体中都有报道其活性，但这两种细胞器中酶的相对含量尚不清楚。亚铁螯合酶难以测定，因为亚铁需要严格的厌氧条件来防止氧化，并且在光合组织中叶绿素干扰产物的定量。因此，我们开发了一种灵敏的荧光测定亚铁螯合酶，采用钴+和次卟啉代替天然底物，并测量次卟啉荧光的减少。对于绿色组织，包括去除叶绿素的己烷提取步骤。该测定在叶绿体蛋白浓度范围内呈线性，平均比活度为0.68 nmol。min（-1）。毫克蛋白质（-1），最高的报告。线粒体的相应值为0.19 nmol。min（-1）。蛋白质（-1）该酶被N-甲基原卟啉抑制，估计IC 50值约为1 nM。使用该测定法，我们已经定量了来自绿色豌豆叶、黄化豌豆叶和豌豆根的质体和线粒体中的亚铁螯合酶活性，以确定两个细胞器中的相对量。我们发现，在所有三种组织中，超过90%。的活性与质体有关，但亚铁螯合酶在线粒体中可重复检测到，其水平高于污染质体标记酶，并且是潜伏的。我们的研究结果表明，质体是高等植物细胞中血红素生物合成的主要场所，但线粒体也具有血红素生产的能力。
Ferrochelatase is the terminal enzyme of haem biosynthesis, catalysing the insertion of ferrous iron into the macrocycle of protoporphyrin IX, the last common intermediate of haem and chlorophyll synthesis. Its activity has been reported in both plastids and mitochondria of higher plants, but the relative amounts of the enzyme in the two organelles are unknown. Ferrochelatase is difficult to assay since ferrous iron requires strict anaerobic conditions to prevent oxidation, and in photosynthetic tissues chlorophyll interferes with the quantification of the product. Accordingly, we developed a sensitive fluorimetric assay for ferrochelatase that employs Co2+ and deuteroporphyrin in place of the natural substrates, and measures the decrease in deuteroporphyrin fluorescence. A hexane-extraction step to remove chlorophyll is included for green tissue. The assay is linear over a range of chloroplast protein concentrations, with an average specific activity of 0.68 nmol . min(-1) . mg of protein(-1), the highest yet reported. The corresponding value for mitochondria is 0.19 nmol . min(-1) . mg of protein(-1). The enzyme is inhibited by N-methylprotoporphyrin, with an estimated IC50 value of approximate to 1 nM. Using this assay we have quantified ferrochelatase activity in plastids and mitochondria from green pea leaves, etiolated pea leaves and pea roots to determine the relative amounts in the two organelles. We found that, in all three tissues, greater than 90%. of the activity was associated with plastids, but ferrochelatase was reproducibly detected in mitochondria, at levels greater than the contaminating plastid marker enzyme, and was latent. Our results indicate that plastids are the major site of haem biosynthesis in higher plant cells, but that mitochondria also have the capacity for haem production.