嗜酸氧化亚铁硫杆菌铁硫簇生物合成机理研究
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摘要
生物冶金技术是一种利用微生物从矿物中提取有价金属的经济方法,具有成本低、投入少、能耗低,污染小等突出优点；针对我国有色矿产资源低品位、复杂、难处理的特点,生物冶金作为一种矿产资源高效提取的新技术具有广阔的应用前景。嗜酸氧化亚铁硫杆菌是最早发现和应用于微生物冶金的细菌,在微生物冶金中扮演极其重要角色,其作为硫化矿生物浸出机理研究的模式菌株一直是研究的热点。
本论文以嗜酸氧化亚铁硫杆菌A.ferrooxidans ATCC23270为模板,研究了在其能量代谢过程中电子传递途径里起重要作用的铁硫簇的合成及转运机制。以嗜酸氧化亚铁硫杆菌的ISC铁硫簇系统为研究对象,克隆表达了ISC铁硫簇生物合成系统的三个核心蛋白：半胱氨酸脱硫酶蛋白IscS,铁硫簇组装的支架蛋白IscU以及另一个铁硫簇组装的支架蛋白/铁结合蛋白IscA。分别研究了它们在铁硫簇生物合成过程中的功能和作用机理,并探讨了它们在铁硫簇的生物合成和传递过程中相互作用机制,以及不同氧化应激条件下铁硫簇生物合成的差异表达；同时研究了嗜酸氧化亚铁硫杆菌硫代谢过程中催化硫酸盐发生首步同化反应的ATP硫酸化酶CysD1,进一步加深了对嗜酸氧化亚铁硫杆菌调控整个硫代谢途径从硫酸盐到半胱氨酸的电子传递过程cys操纵子的理解。希望通过这些工作为加深对嗜酸氧化亚铁硫杆菌铁硫代谢途径及电子传递机理的了解提供理论基础。
本文的研究工作主要包括以下几个方面：
(1)克隆了嗜酸氧化亚铁硫杆菌ATCC23270的isc操纵子中表达支架蛋白的IscU基因,并转入了含高效T7启动子的pET28b表达质粒。成功地在大肠杆菌BL21(DE3)中表达了IscU,并对蛋白诱导条件进行了优化。发现在IPTG浓度为0.5mM,室温过夜的诱导条件下能得到较高水平重组IscU的表达。表达后的重组蛋白主要以可溶性的形式存在于细胞上清中,应用饱和的鳌合型硫酸镍琼脂糖树脂柱对含组氨酸标签的IscU蛋白进行了纯化,得到分子量大小为16kD的IscU蛋白,每升LB培养液可获得6mg IscU蛋白。从大肠杆菌纯化得到的IscU蛋白呈无色透明状,自身不带有铁硫簇结构；铁结合实验发现IscU蛋白体外和体内均不具备结合铁的能力；IscU可以作为支架蛋白完成铁硫簇的组装,在体外添加外源铁离子和硫离子能够在20min内完成apo-IscU到[2Fe-2S]-IscU铁硫簇的组装。pH值对IscU上铁硫簇的生物合成具有明显的作用,在pH7.0-8.0时IscU可以较好的完成自身铁硫簇的生物组装,而当反应条件从pH7.0降低到6.0时,IscU上铁硫簇的生物合成受到极大的抑制。而当pH值小于6.0时,体系中蛋白发生变性出现沉淀,无法完成铁硫簇的组装。
(2)克隆了嗜酸氧化亚铁硫杆菌ATCC23270的isc操纵子中表达半胱氨酸脱硫酶的IscS基因,转入了含T7高效启动子的pET28b表达质粒。成功地在大肠杆菌BL21(DE3)中表达了IscS,每1升LB培养液可表达并纯化获得5.2mg IscS蛋白。经纯化之后的IscS蛋白呈亮黄色,蛋白分子量为46kD。对纯化的IscS蛋白进行酶活测定,发现IscS具有较强的半胱氨酸脱硫酶活性,能催化L-半胱氨酸脱硫并把硫释放到溶液中。加入磷酸吡哆醛能促进IscS的酶活的表达。在温度为20-40℃之时IscS表现出较高的酶活性,其最适反应温度为37℃。而当反应温度低于20℃或者高于40℃时都将极大的抑制IscS的酶活性；pH8.0为IscS酶活的最适反应条件,在pH7.0和9.0之间IscS具有较强的酶活性,而超出这个范围将极大的削弱IscS的酶活。IscS在铁硫簇的体内和体外合成中都具有重要的作用,IscS可以催化L-半胱氨酸为apo-fdx体外组装[2Fe-2S]-fdx的铁硫簇组装提供硫源。在LB培养基中加入IscS酶活抑制剂L-烯丙基甘氨酸将极大的抑制IscS酶活的表达,从而阻断[2Fe-2S]-fdx体内的表达。IscS能和铁离子,半胱氨酸结合形成IscS-iron和IscS-iron-sulfur的复合物,复合物的存在将会抑制IscS半胱氨酸脱硫酶的活性。IscS的酶活受到反应体系中铁离子和硫离子的反馈调节作用对于铁硫簇生物合成机制的具有重要的作用。
(3)克隆了嗜酸氧化亚铁硫杆菌ATCC23270的isc操纵子中的IscA基因,并构建了pET28b-IscA表达质粒。成功地在大肠杆菌BL21(DE3)中表达了IscA,每1升LB培养液可表达并纯化获得7.0mg IscA蛋白。经纯化之后的IscA蛋白呈亮黄色,蛋白分子量为11kD。从LB培养基中纯化得到的IscA是一个含4Fe-4S]铁硫簇的铁硫蛋白,加入EDTA和L-半胱氨酸处理后可以除去[4Fe-4S]簇形成apo-IscA。Apo-IscA在体外具有较强的铁的结合活性,可以结合铁离子形成iron-loaded IscA;而iron-loaded IscA可以在IscS (?)L-半胱氨酸存在的情况下在体外转化成[4Fe-4S]-IscA。以上apo-IscA, iron-loaded IscA和[4Fe-4S]-IscA三个不同状态的IscA代表了铁硫簇在IscA上合成时三个不同的阶段。IscA蛋白中Cys35, Cys99和Cys101是其结合铁和铁硫簇的关键位点,对这三个关键氨基酸位点进行定点突变成丙氨酸后的IscA蛋白失去了结合铁和铁硫簇的能力。IscA对三价铁具有较强的铁结合活性,氧气可以在IscA的铁结合位点上通过氧化二价铁成三价铁而促进了IscA对铁的结合作用。同时Iron-loaded IscA可以作为铁供体参与支架蛋白IscU上铁硫簇的生物组装。
(4)支架蛋白IscU在低铁离子浓度下无法完成自身铁硫簇的生物组装,而IscA可以竞争性的结合反应体系中的铁离子,并将结合的铁离子传递给IscU完成IscU上铁硫簇的组装。L-半胱氨酸可以修饰iron-loaded IscA中铁的活性中心,使铁离子得以释放到反应体系中。同时L-半胱氨酸可以作为IscS酶活反应的底物脱硫产生硫离子,而IscU可以结合iron-loaded IscA中被L-半胱氨酸修饰的铁离子以及L-半胱氨酸脱硫产生的硫离子,完成[2Fe-2S]铁硫簇的组装。对嗜酸氧化亚铁硫杆菌isc操纵子中的IscU和IscA在铁硫簇生物合成过程中的相互作用研究发现,IscU是一个优先被选择的铁硫簇组装的支架蛋白,而高铁结合活性的IscA是一个优先被选择的铁结合蛋白。含有铁硫簇的支架蛋白可以把自身的铁硫簇传递给apo形式的铁硫蛋白。带[4Fe-4S]铁硫簇的holo-IscA可以把自身的铁硫簇传递给apo-fdx和apo-APS reductase完成[2Fe-2S]-fdx和[4Fe-4S]-APS reductase的组装,完成铁硫簇传递后的holo-fdx和holo-APS reductase的铁硫蛋白具有完整的生物学活性。
(5)克隆了嗜酸氧化亚铁硫杆菌ATCC23270的硫代谢途径cys操纵子中编码ATP硫酸化酶的CysD1基因,并构建了pET28b-CysD1表达质粒。成功地在大肠杆菌BL21(DE3)中表达了CysD1,每1升LB培养液可表达并纯化获得4.3mgCysD1蛋白。经纯化之后的CysD1蛋白透明无色,蛋白分子量为33kD。从LB培养基中纯化得到的CysD1紫外扫描在420nm处存在一个特征吸收峰,具有催化硫酸根离子和ATP生成APS和焦磷酸的能力,采用生物发光法测定其ATP硫酸化酶的活性为3.0×103U/mg。对CysD1蛋白序列进行同源性比对,结合蛋白三维结构的模拟,采用蛋白定点突变,将170位Gln,173位的Arg和211位的Asp分别突变为Ser后,紫外扫描发现CysD1在420nm处的特征吸收峰消失,同时也极大的削弱了其ATP硫酸化酶催化硫酸根离子和ATP生成APS和焦磷酸的酶活能力,说明Q170,R173和D211这三个关键位点是嗜酸氧化亚铁硫杆菌ATCC23270的ATP硫酸化酶结合ADP活性的关键位点。
Acidithiobacillus ferrooxidans is a widely studied bacterium that lives in acid mine drainage, which obtains energy through the oxidation of ferrous ion to ferric ion with O2as the terminal electron acceptor. Acidithiobacillus ferrooxidans has attracted much attention in studying the mechanism of bioleaching. Iron sulfur clusters are one of the most ancient and ubiquitous redox centre in almost all living organism and play an important role in photosynthesis, respiration and nitrogen fixation. In this study, we usd the Acidithiobacillus ferrooxidans ATCC23270as the type strain to study the biogenesis and transfer mechanism of iron-sulfur clusters. The primary subjects of which include six parts as follows:
(1) We cloned the gene of iron-sulfur cluster assembly scaffold protein IscU from the isc operon and constituted the plasmid pET28b-IscU. The recombinant plasmid was successfully expressed in LB medium with0.5mM IPTG under the room temperature overnight. The recombinant IscU protein was expressed as fusion proteins with a His-tag and purified by affinity chromatography using Ni-NTA agarose gel attached with the AKTA explore. IscU had a molecular weight of16kD and are colourless. The purified IscU did not have any iron-sulfur cluster and could not bind iron neither in vitro nor in vivo. IscU colud act as a scaffold protein to assemble the [2Fe-2S] cluster within30min. pH is an important impact factor for biogenesis of iron-sulfur cluster in IscU, the suitable pH value for iron sulfur cluster assemble is pH7.0-8.0, decreasing the pH value will restrain the iron-sulfur cluster assembly.
(2) The gene of cysteine desulfurase protein iscS from the isc operon was cloned and constituted the into the plasmid pET28b. The recombinant plasmid pET28b-IscS was successfully expressed in LB medium and purified by affinity chromatography. IscS had a molecular weight of46kD and was light yellow in color. The purified IscS had high cysteine desulfurase activity, could catalyze desulfurization of L-cysteine. Incubating IscS with PLP would greatly increase the activity of IscS. The most suitable condition for IscS cysteine desulfurase activity was37℃and pH8.0. IscS was very important for iron-sulfur cluster assembly both in vitro and in vivo, it could catalyze desulfurization of L-cysteine and transfer sulfur for iron-sulfur cluster assembly on apo-fdx in vitro, inactivation of IscS serious affected the iron-sulfur cluster assembly on fdx in vivo.
(3) The gene of IscA from A. ferrooxidans ATCC23270was cloned, expressed and purified by affinity chromatography. The purified IscA had a molecular weight of11kD and was brown in color. Acidithiobacillus ferrooxidans IscA is a [4Fe-4S] cluster binding protein, and it can bind iron in the presence of DTT with an apparent iron association constant of4×1020M-1. The iron binding in IscA can be promoted by oxygen through oxidizing ferrous iron to ferric iron. Furthermore, we show that the iron bound form IscA can be converted to iron sulfur cluster bound form in the presence of IscS and L-cysteine in vitro. Substitution of the invariant cysteine residues Cys35, Cys99or Cys101in IscA abolishes the iron binding activity of the protein; the IscA mutants that fail to bind iron are unable to assemble the iron sulfur clusters. Further studies indicate that the iron-loaded IscA could act as an iron donor for the assembly of iron-sulfur clusters in the scaffold protein IscU in vitro.
(4) L-cysteine could mobilize the iron centre in IscA and to provide sulfur via IscS for iron-sulfur cluster assembly in IscU. When equal amounts of IscA and IscU are incubated with ferrous iron in the presence of IscS, L-cysteine and DTT, iron-sulfur cluster are assembled in IscU, suggested IscU is a preferred iron-sulfur cluster assembly scaffold protein. In contract, when equal amounts of IscA and IscU are incubated with ferrous iron in the presence of IscS and DTT, nearly all iorn is bound to IscA, suggested IscA is a preferred iron binding protein. Iron sulfur clusters could be transfer from pre-assembled scaffold proteins to apo-form iron sulfur proteins. Holo-IscA could transfer its [4Fe-4S] cluster to apo-fdx and apo-APS reductase to form [2Fe-2S]-fdx and [4Fe-4S]-APS reductase. After the iron-sulfur cluster transferring, the reconstitution iron sulfur proteins could restore their physiological activities.
(5) The gene of ATP sulfurylase cysDl from the cys operon was cloned and constituted the into the plasmid pET28b. The recombinant plasmid pET28b-CysDl was successfully expressed in LB medium and purified by affinity chromatography. CysDl had a molecular weight of33kD and was colorless. The purified ATP sulfurylase could catalysis ATP and sulfate to form APS and pyrophosphate, the specific activity of the purified ATP sulfurylase was3.0×103U/mg. We constructed the mutant expression plasmid (Q170S, R173S, D211S) of the protein using the Site-directed mutagenesis. Molecular modelling for the protein, UV-Vis scanning and enzyme activity revealed that Q170, R173and D211were curcial sites for ATP sulfurylase bindings.
本论文以嗜酸氧化亚铁硫杆菌A.ferrooxidans ATCC23270为模板,研究了在其能量代谢过程中电子传递途径里起重要作用的铁硫簇的合成及转运机制。以嗜酸氧化亚铁硫杆菌的ISC铁硫簇系统为研究对象,克隆表达了ISC铁硫簇生物合成系统的三个核心蛋白：半胱氨酸脱硫酶蛋白IscS,铁硫簇组装的支架蛋白IscU以及另一个铁硫簇组装的支架蛋白/铁结合蛋白IscA。分别研究了它们在铁硫簇生物合成过程中的功能和作用机理,并探讨了它们在铁硫簇的生物合成和传递过程中相互作用机制,以及不同氧化应激条件下铁硫簇生物合成的差异表达；同时研究了嗜酸氧化亚铁硫杆菌硫代谢过程中催化硫酸盐发生首步同化反应的ATP硫酸化酶CysD1,进一步加深了对嗜酸氧化亚铁硫杆菌调控整个硫代谢途径从硫酸盐到半胱氨酸的电子传递过程cys操纵子的理解。希望通过这些工作为加深对嗜酸氧化亚铁硫杆菌铁硫代谢途径及电子传递机理的了解提供理论基础。
本文的研究工作主要包括以下几个方面：
(1)克隆了嗜酸氧化亚铁硫杆菌ATCC23270的isc操纵子中表达支架蛋白的IscU基因,并转入了含高效T7启动子的pET28b表达质粒。成功地在大肠杆菌BL21(DE3)中表达了IscU,并对蛋白诱导条件进行了优化。发现在IPTG浓度为0.5mM,室温过夜的诱导条件下能得到较高水平重组IscU的表达。表达后的重组蛋白主要以可溶性的形式存在于细胞上清中,应用饱和的鳌合型硫酸镍琼脂糖树脂柱对含组氨酸标签的IscU蛋白进行了纯化,得到分子量大小为16kD的IscU蛋白,每升LB培养液可获得6mg IscU蛋白。从大肠杆菌纯化得到的IscU蛋白呈无色透明状,自身不带有铁硫簇结构；铁结合实验发现IscU蛋白体外和体内均不具备结合铁的能力；IscU可以作为支架蛋白完成铁硫簇的组装,在体外添加外源铁离子和硫离子能够在20min内完成apo-IscU到[2Fe-2S]-IscU铁硫簇的组装。pH值对IscU上铁硫簇的生物合成具有明显的作用,在pH7.0-8.0时IscU可以较好的完成自身铁硫簇的生物组装,而当反应条件从pH7.0降低到6.0时,IscU上铁硫簇的生物合成受到极大的抑制。而当pH值小于6.0时,体系中蛋白发生变性出现沉淀,无法完成铁硫簇的组装。
(2)克隆了嗜酸氧化亚铁硫杆菌ATCC23270的isc操纵子中表达半胱氨酸脱硫酶的IscS基因,转入了含T7高效启动子的pET28b表达质粒。成功地在大肠杆菌BL21(DE3)中表达了IscS,每1升LB培养液可表达并纯化获得5.2mg IscS蛋白。经纯化之后的IscS蛋白呈亮黄色,蛋白分子量为46kD。对纯化的IscS蛋白进行酶活测定,发现IscS具有较强的半胱氨酸脱硫酶活性,能催化L-半胱氨酸脱硫并把硫释放到溶液中。加入磷酸吡哆醛能促进IscS的酶活的表达。在温度为20-40℃之时IscS表现出较高的酶活性,其最适反应温度为37℃。而当反应温度低于20℃或者高于40℃时都将极大的抑制IscS的酶活性；pH8.0为IscS酶活的最适反应条件,在pH7.0和9.0之间IscS具有较强的酶活性,而超出这个范围将极大的削弱IscS的酶活。IscS在铁硫簇的体内和体外合成中都具有重要的作用,IscS可以催化L-半胱氨酸为apo-fdx体外组装[2Fe-2S]-fdx的铁硫簇组装提供硫源。在LB培养基中加入IscS酶活抑制剂L-烯丙基甘氨酸将极大的抑制IscS酶活的表达,从而阻断[2Fe-2S]-fdx体内的表达。IscS能和铁离子,半胱氨酸结合形成IscS-iron和IscS-iron-sulfur的复合物,复合物的存在将会抑制IscS半胱氨酸脱硫酶的活性。IscS的酶活受到反应体系中铁离子和硫离子的反馈调节作用对于铁硫簇生物合成机制的具有重要的作用。
(3)克隆了嗜酸氧化亚铁硫杆菌ATCC23270的isc操纵子中的IscA基因,并构建了pET28b-IscA表达质粒。成功地在大肠杆菌BL21(DE3)中表达了IscA,每1升LB培养液可表达并纯化获得7.0mg IscA蛋白。经纯化之后的IscA蛋白呈亮黄色,蛋白分子量为11kD。从LB培养基中纯化得到的IscA是一个含4Fe-4S]铁硫簇的铁硫蛋白,加入EDTA和L-半胱氨酸处理后可以除去[4Fe-4S]簇形成apo-IscA。Apo-IscA在体外具有较强的铁的结合活性,可以结合铁离子形成iron-loaded IscA;而iron-loaded IscA可以在IscS (?)L-半胱氨酸存在的情况下在体外转化成[4Fe-4S]-IscA。以上apo-IscA, iron-loaded IscA和[4Fe-4S]-IscA三个不同状态的IscA代表了铁硫簇在IscA上合成时三个不同的阶段。IscA蛋白中Cys35, Cys99和Cys101是其结合铁和铁硫簇的关键位点,对这三个关键氨基酸位点进行定点突变成丙氨酸后的IscA蛋白失去了结合铁和铁硫簇的能力。IscA对三价铁具有较强的铁结合活性,氧气可以在IscA的铁结合位点上通过氧化二价铁成三价铁而促进了IscA对铁的结合作用。同时Iron-loaded IscA可以作为铁供体参与支架蛋白IscU上铁硫簇的生物组装。
(4)支架蛋白IscU在低铁离子浓度下无法完成自身铁硫簇的生物组装,而IscA可以竞争性的结合反应体系中的铁离子,并将结合的铁离子传递给IscU完成IscU上铁硫簇的组装。L-半胱氨酸可以修饰iron-loaded IscA中铁的活性中心,使铁离子得以释放到反应体系中。同时L-半胱氨酸可以作为IscS酶活反应的底物脱硫产生硫离子,而IscU可以结合iron-loaded IscA中被L-半胱氨酸修饰的铁离子以及L-半胱氨酸脱硫产生的硫离子,完成[2Fe-2S]铁硫簇的组装。对嗜酸氧化亚铁硫杆菌isc操纵子中的IscU和IscA在铁硫簇生物合成过程中的相互作用研究发现,IscU是一个优先被选择的铁硫簇组装的支架蛋白,而高铁结合活性的IscA是一个优先被选择的铁结合蛋白。含有铁硫簇的支架蛋白可以把自身的铁硫簇传递给apo形式的铁硫蛋白。带[4Fe-4S]铁硫簇的holo-IscA可以把自身的铁硫簇传递给apo-fdx和apo-APS reductase完成[2Fe-2S]-fdx和[4Fe-4S]-APS reductase的组装,完成铁硫簇传递后的holo-fdx和holo-APS reductase的铁硫蛋白具有完整的生物学活性。
(5)克隆了嗜酸氧化亚铁硫杆菌ATCC23270的硫代谢途径cys操纵子中编码ATP硫酸化酶的CysD1基因,并构建了pET28b-CysD1表达质粒。成功地在大肠杆菌BL21(DE3)中表达了CysD1,每1升LB培养液可表达并纯化获得4.3mgCysD1蛋白。经纯化之后的CysD1蛋白透明无色,蛋白分子量为33kD。从LB培养基中纯化得到的CysD1紫外扫描在420nm处存在一个特征吸收峰,具有催化硫酸根离子和ATP生成APS和焦磷酸的能力,采用生物发光法测定其ATP硫酸化酶的活性为3.0×103U/mg。对CysD1蛋白序列进行同源性比对,结合蛋白三维结构的模拟,采用蛋白定点突变,将170位Gln,173位的Arg和211位的Asp分别突变为Ser后,紫外扫描发现CysD1在420nm处的特征吸收峰消失,同时也极大的削弱了其ATP硫酸化酶催化硫酸根离子和ATP生成APS和焦磷酸的酶活能力,说明Q170,R173和D211这三个关键位点是嗜酸氧化亚铁硫杆菌ATCC23270的ATP硫酸化酶结合ADP活性的关键位点。
Acidithiobacillus ferrooxidans is a widely studied bacterium that lives in acid mine drainage, which obtains energy through the oxidation of ferrous ion to ferric ion with O2as the terminal electron acceptor. Acidithiobacillus ferrooxidans has attracted much attention in studying the mechanism of bioleaching. Iron sulfur clusters are one of the most ancient and ubiquitous redox centre in almost all living organism and play an important role in photosynthesis, respiration and nitrogen fixation. In this study, we usd the Acidithiobacillus ferrooxidans ATCC23270as the type strain to study the biogenesis and transfer mechanism of iron-sulfur clusters. The primary subjects of which include six parts as follows:
(1) We cloned the gene of iron-sulfur cluster assembly scaffold protein IscU from the isc operon and constituted the plasmid pET28b-IscU. The recombinant plasmid was successfully expressed in LB medium with0.5mM IPTG under the room temperature overnight. The recombinant IscU protein was expressed as fusion proteins with a His-tag and purified by affinity chromatography using Ni-NTA agarose gel attached with the AKTA explore. IscU had a molecular weight of16kD and are colourless. The purified IscU did not have any iron-sulfur cluster and could not bind iron neither in vitro nor in vivo. IscU colud act as a scaffold protein to assemble the [2Fe-2S] cluster within30min. pH is an important impact factor for biogenesis of iron-sulfur cluster in IscU, the suitable pH value for iron sulfur cluster assemble is pH7.0-8.0, decreasing the pH value will restrain the iron-sulfur cluster assembly.
(2) The gene of cysteine desulfurase protein iscS from the isc operon was cloned and constituted the into the plasmid pET28b. The recombinant plasmid pET28b-IscS was successfully expressed in LB medium and purified by affinity chromatography. IscS had a molecular weight of46kD and was light yellow in color. The purified IscS had high cysteine desulfurase activity, could catalyze desulfurization of L-cysteine. Incubating IscS with PLP would greatly increase the activity of IscS. The most suitable condition for IscS cysteine desulfurase activity was37℃and pH8.0. IscS was very important for iron-sulfur cluster assembly both in vitro and in vivo, it could catalyze desulfurization of L-cysteine and transfer sulfur for iron-sulfur cluster assembly on apo-fdx in vitro, inactivation of IscS serious affected the iron-sulfur cluster assembly on fdx in vivo.
(3) The gene of IscA from A. ferrooxidans ATCC23270was cloned, expressed and purified by affinity chromatography. The purified IscA had a molecular weight of11kD and was brown in color. Acidithiobacillus ferrooxidans IscA is a [4Fe-4S] cluster binding protein, and it can bind iron in the presence of DTT with an apparent iron association constant of4×1020M-1. The iron binding in IscA can be promoted by oxygen through oxidizing ferrous iron to ferric iron. Furthermore, we show that the iron bound form IscA can be converted to iron sulfur cluster bound form in the presence of IscS and L-cysteine in vitro. Substitution of the invariant cysteine residues Cys35, Cys99or Cys101in IscA abolishes the iron binding activity of the protein; the IscA mutants that fail to bind iron are unable to assemble the iron sulfur clusters. Further studies indicate that the iron-loaded IscA could act as an iron donor for the assembly of iron-sulfur clusters in the scaffold protein IscU in vitro.
(4) L-cysteine could mobilize the iron centre in IscA and to provide sulfur via IscS for iron-sulfur cluster assembly in IscU. When equal amounts of IscA and IscU are incubated with ferrous iron in the presence of IscS, L-cysteine and DTT, iron-sulfur cluster are assembled in IscU, suggested IscU is a preferred iron-sulfur cluster assembly scaffold protein. In contract, when equal amounts of IscA and IscU are incubated with ferrous iron in the presence of IscS and DTT, nearly all iorn is bound to IscA, suggested IscA is a preferred iron binding protein. Iron sulfur clusters could be transfer from pre-assembled scaffold proteins to apo-form iron sulfur proteins. Holo-IscA could transfer its [4Fe-4S] cluster to apo-fdx and apo-APS reductase to form [2Fe-2S]-fdx and [4Fe-4S]-APS reductase. After the iron-sulfur cluster transferring, the reconstitution iron sulfur proteins could restore their physiological activities.
(5) The gene of ATP sulfurylase cysDl from the cys operon was cloned and constituted the into the plasmid pET28b. The recombinant plasmid pET28b-CysDl was successfully expressed in LB medium and purified by affinity chromatography. CysDl had a molecular weight of33kD and was colorless. The purified ATP sulfurylase could catalysis ATP and sulfate to form APS and pyrophosphate, the specific activity of the purified ATP sulfurylase was3.0×103U/mg. We constructed the mutant expression plasmid (Q170S, R173S, D211S) of the protein using the Site-directed mutagenesis. Molecular modelling for the protein, UV-Vis scanning and enzyme activity revealed that Q170, R173and D211were curcial sites for ATP sulfurylase bindings.
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