HUMAN FERROCHELATASE IS AN IRON-SULFUR PROTEIN
HUMAN FERROCHELATASE IS AN IRON-SULFUR PROTEIN
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DOI：
10.1021/bi00168a003
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发表时间：
1994-01-18
期刊：
影响因子：
2.9
通讯作者：
JOHNSON, MK
中科院分区：
文献类型：
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作者：
DAILEY, HA;FINNEGAN, MG;JOHNSON, MK
重组人亚铁螯合酶已在大肠杆菌中表达并纯化至均一。金属分析显示几乎等于2摩尔的非血红素铁每摩尔的纯化的酶（M（r）= 40 000）。纯化酶的紫外-可见吸收光谱由278 nm处的蛋白吸收组成（几乎等于90 000 M-1 cm-1）和330 nm处的条带（几乎等于24 000 M-1 cm-1），460 nm（肩峰，肩峰几乎等于11000 M-1cm-1）和550 nm（肩峰，肩峰几乎等于9000 M-1cm-1）的特征表明存在[2Fe-2S]2+簇。光谱显示在415 nm处的一个额外的带，不同的制剂的强度不同，至少部分归因于酶相关的高自旋Fe（III）血红素的次要成分。一个单一的[2Fe-2S]2+，+集群作为人亚铁螯合酶的氧化还原活性成分的存在下，通过变温MCD和EPR研究的连二亚硫酸盐还原的酶，这表明存在的S = 1/2 [2Fe-2S]+集群除了残留的高自旋Fe（II）血红素。还原的酶表现出S = 1/2 EPR信号，g = 2.00，1.94，1.91，占0.75 +/- 0.25自旋/分子，在低于10 K的低微波功率下容易饱和，但在高达100 K的温度下可观察到没有显著的增宽。当酶在4 ℃有氧储存时，Fe-S簇是不稳定的，并在24小时内逐渐消失，伴随着酶活性的丧失。基因工程改造的C-末端截短的人铁螯合酶，以及正常的酵母和E。coli铁螯合酶缺乏[2Fe-2S]簇。这些数据表明，人亚铁螯合酶含有一个[2Fe-2S]2+，+集群，这是必不可少的活动，并协调至少一个和可能所有的四个半胱氨酸是保守的哺乳动物亚铁螯合酶的C-末端区域。
Recombinant human ferrochelatase has been expressed in Escherichia coli and purified to homogeneity. Metal analyses revealed almost-equal-to 2 mol of non-heme Fe per mol of the purified enzyme (M(r) = 40 000). The UV-visible absorption spectrum of the purified enzyme consists of a protein absorption at 278 nm (epsilon almost-equal-to 90 000 M-1 cm-1) and bands at 330 nm (epsilon almost-equal-to 24 000 M-1 cm-1), 460 nm (shoulder, epsilon almost-equal-to 11 000 M-1 cm-1), and 550 nm (shoulder, epsilon almost-equal-to 9000 M-1 cm-1) that are indicative of a [2Fe-2S]2+ cluster. The spectra show an additional band at 415 nm that varied in intensity for different preparations and is attributed, at least in part, to a minor component of enzyme-associated high-spin Fe(III) heme. The presence of a single [2Fe-2S]2+,+ Cluster as a redox active component of human ferrochelatase was confirmed by variable-temperature MCD and EPR studies of the dithionite-reduced enzyme which showed the presence of a S = 1/2 [2Fe-2S]+ cluster in addition to residual high spin Fe(II) heme. The reduced enzyme exhibits a S = 1/2 EPR signal, g = 2.00, 1.94, 1.91 accounting for 0.75 +/- 0.25 spins/molecule, that readily saturates at low microwave powers below 10 K but is observable without significant broadening at temperatures up to 100 K. The Fe-S cluster is labile and gradually disappears over period of 24 h, with concomitant loss of enzyme activity, when the enzyme is stored aerobically at 4-degrees-C. Genetically engineered C-terminal truncated human ferrochelatase, as well as the normal yeast and E. coli ferrochelatases, lack the [2Fe-2S] cluster. These data suggest that human ferrochelatase contains a [2Fe-2S]2+,+ cluster that is essential for activity and is coordinated by at least one and probably all of the four cysteines that are conserved in the C-terminal region of mammalian ferrochelatases.