【作者】 刘元东；

【导师】 邱冠周；

【作者基本信息】 中南大学 ， 生物工程， 2008， 博士

【摘要】 环境友好高效的生物冶金技术的工业应用和发展亟需理论的突破以给生产实践提供指导,但由于对生物因素了解的局限性及生物冶金体系本身的复杂性一直妨碍了人们对生物冶金体系的全局的理解。本论文运用序列比对与识别、同源模建、进化踪迹分析、途径网络重构、分子模拟、量子化学计算、静电势分析、分子对接等生物信息学和分子计算方法,结合蛋白质表达纯化和定点突变与表征等技术,对嗜酸氧化亚铁硫杆菌（Acidithiobacillus ferrooxidans,简称A.f菌）在浸矿过程中利用铁硫能源驱动生物体合成的铁硫代谢能量传递过程进行了系统地研究,构建了其能量传递途径网络图,解释了单个A.f菌如何浸矿。此外,对硫化矿生物浸出体系及其能量传递耦合的产酸问题进行了分析。论文的具体研究内容和结果可概括为五个方面:一.A.f菌的若干能量传递蛋白的结构分析与反应机理预测。细胞色素c:A.f菌有10个细胞色素c,聚类分析表明它们分成4大同源蛋白类。其中4个周质细胞色素c₄家族蛋白的序列结构比较表明位于Cyc1的Tyr63和Glu121位点附近的残基差异决定它们的功能差异。细胞色素bc₁复合体:A.f菌有2个细胞色素bc₁复合体,对它们进行了结构模建和机理探讨,序列和结构比较显示了它们在细胞色素b亚基位于血红素b_L辅基近溶液外侧的Arg79位点存在差异,这可能导致了它们的正反向功能差异。终端氧化酶:A.f菌有4个终端氧化酶,对它们进行了结构模建和机理探讨,结果表明,细胞色素aa₃和ba₃复合体的电子供体是周质细胞色素c;细胞色素bo₃和bd复合体的电子供体是泛醌。细胞色素CycA1:模建了三维分子结构,它包含2个血红素辅基。细胞色素Cyc2_like蛋白:生物信息学分析表明它是定位于外膜含一个血红素的高分子量细胞色素蛋白。硫化物泛醌还原酶（SQR）:SQR的结构尚未解析,模建了A.f菌源SQR三维分子结构,并跟黄素（FAD）和泛醌（UQ）进行分子对接,根据这些结果提出了其反应机理。基因doxDA:A.f菌doxDA-1与doxDA-2基因的生物信息学分析表明它们是融合基因。DsbG蛋白:结构模建结果表明,二聚体DsbG蛋白中的两个单体对称结合,2个保守的Cys119和Cys122残基的位置远离二聚体DsbG蛋白的接触面,但它们的位置十分接近。核糖-5-磷酸异构酶:模建了三维分子结构并跟底物R5P进行了分子对接,根据对接结果识别了催化的关键残基,提出了其反应机理。谷胱甘肽还原酶:模建了三维分子结构并跟底物进行分子对接,根据对接结果识别了催化的关键残基,提出了其反应机理。二.A.f菌的若干能量传递蛋白的结构模建与表达验证。铜蓝蛋白:静电势分析、活性位点量化计算和蛋白质表达定点突变实验证实残基Cys138对结合铜原子非常重要。亚铁氧化酶（Iro）:Iro的结构尚未解析,模建了A.f菌源Iro结构,结果表明它是由4个半胱氨酸配位[Fe₄S₄]铁硫簇的高电位铁硫蛋白（HiPIP）家族蛋白,对离铁硫簇十分近的Tyr10虚拟突变实验预测了该位置处的芳香环对保护铁硫簇起重大作用,蛋白质的表达纯化及定点突变实验进一步证实了这些结论。高电位铁硫蛋白:结构模建表明它含有4个半胱氨酸配位的[Fe₄S₄]铁硫簇;蛋白的表达纯化实验表明它含有铁硫簇活性中心;定点突变实验进一步证实预测的残基配位[Fe₄S₄]铁硫簇。亚硫酸还原酶:模建了它的黄素蛋白（SiR-FP）和血红素蛋白（SiR-HP）的三维分子结构,结果表明,SiR-HP中4个保守的Cys427,Cys433,Cys472和Cys476残基配位位于单体结构的中心的[Fe₄S₄]铁硫簇,血红素位于铁硫簇附近。蛋白的表达纯化实验表明SiR-FP含有[Fe₄S₄]铁硫簇活性中心和血红素基团,定点突变实验证实预测的残基配位[Fe₄S₄]铁硫簇。APS还原酶:结构模建结果表明它含有由4个保守的Cys110,Cys111,Cys193和Cys196残基配位[Fe₄S₄]铁硫簇,底物APS分子对接的结果显示其位置就在附近。蛋白的表达纯化实验表明它含有铁硫簇活性中心。铁硫簇组装供硫蛋白（IscS）:模建了A.f菌源IscS的完整的三维分子结构,跟辅因子PLP和底物半胱氨酸分子对接发现了一些重要残基;蛋白的表达纯化实验进一步证勃A.f菌源的IscS是一个半胱氨酸脱硫酶,它依赖PLP,催化L型半胱氨酸转变成L型丙氨酸和硫烷硫,为铁硫簇组装供硫。铁硫簇组装蛋白（IscA）:IscA可能含[Fe₄S₄]或[Fe₂S₂]铁硫簇,结构模建和蛋白的定点突变实验验证表明A.f菌源IscA含有由Cys35,Cys99,Cys101和Glu103配位的[Fe₄S₄]铁硫簇。铁硫簇支架蛋白（IscU）:IscU可能含[Fe₄S₄]或[Fe₂S₂]铁硫簇,结构模建和蛋白的定点突变实验验证表明A.f菌源IscU含有由Cys37,Cys63,Cys106和Asp39配位的[Fe₂S₂]铁硫簇。铁氧还蛋白:铁氧还蛋白可能含[Fe₄S₄],[Fe₃S₄]或[Fe₂S₂]铁硫簇,结构模建表明它是由4个半胱氨酸残基Cys42,Cys48,Cys51和Cys87配位[Fe₂S₂]的铁硫蛋白;蛋白的表达纯化实验表明A.f菌源的铁氧还蛋白正确插入[Fe₂S₂]铁硫簇;定点突变实验进一步证实预测的残基配位[Fe₂S₂]铁硫簇。超氧化物歧化酶:进行了三维结构模建和进化踪迹分析,它的活性中心由Cys35,Cys99,Cys101和Glu103配位铁原子组成;蛋白的表达纯化实验表明它是含铁超氧化物歧化酶。中链乙酰辅酶A脱氢酶:它及其Y375K突变体与乙酰辅酶A的分子对接比较研究和蛋白表达验证结果表明,中链乙酰辅酶A脱氢酶的Tyr375突变成Lys375使其获得了乙酰辅酶A氧化酶活性。乙酰辅酶A氧化酶:分子对接预测和蛋白的表达验证结果表明Oct-2-en-4-ynoyl-CoA是它的一个特异性抑制剂。三.A.f菌的若干多拷贝能量传递蛋白的分子多样性。在三维分子结构层次考察了A.f菌的细胞色素c₄类蛋白、铜蓝蛋白、高氧还电位铁硫蛋白、硫化物泛醌还原酶在不同菌株和相同菌株的不同拷贝间的分子多样性。结果表明相同菌株的不同拷贝蛋白间的差异处于分子的局部;不同菌株同一蛋白间的差异为数个远离活性中心的残基,但有少数在活性中心附近发生突变。四.A.f菌浸矿过程铁硫代谢的能量传递网络图。对A.f ATCC 23270全基因组涉及铁硫代谢能量传递的二百多个基因和三十多个多基因操纵子进行了系统地分析,结合前面各部分的研究结果和已有文献的研究成果,构建了A.f菌浸矿过程铁硫代谢能量传递网络图。它包括矿物的分解,溶液中的反应,A.f菌的呼吸链、亚铁氧化、硫代谢、二氧化碳固定、氮气固定及氢气利用等部分。图中对所涉及蛋白的同工酶和多拷贝蛋白的个数及一些重要反应的氧还电势也进行了标注。通过这个图,可明白单A.f菌如何浸矿。五.硫化矿生物浸出体系的分析及产酸问题。以生成新物质的流量为切入点,系统地分析了硫化矿生物浸出体系,该体系的变化主要由铁硫碳氮氧和金属六条关键物质流主导。最后,根据A.f菌浸矿过程铁硫代谢能量传递网络图结果对硫化矿生物浸出过程能量传递耦合的产酸问题进行了分析。更多还原

【Abstract】 Biohydrometallurgy is an effective technique to extracting metals and friendly to environment.Its industrial application and development need the breakthrough of theory so as to provide guides to the practices. However,the insufficiency for knowing to the biology factor and the complexity for the system of biohydrometallurgy have prevented people from understanding the global of the system of biohydrometallurgy.In this paper,with bioinformatics and molecular computation techniques such as sequence alignment and pattern identification,protein structure building,evolutionary trace analyse,pathway network reconstruction, molecular modeling,quantum chemistry computation,electrostatic potential analyse,molecular docking and so on,combining protein expression,purification,mutation,characterization and so on,the energy transfer process of iron and sulfur metabolism of Acidithiobacillus ferrooxidans utilizing iron and sulfur energy sources to drive the syntheses of its organism in the process of minerals bioleaching was systematically studied,its energy transfer network map was reconstructed. From this map it was explained how A.ferrooxidans to leach minerals. Furthermore,the global analysis of the system of sulfide mineral bioleaching and acid produce coupled by energy transfer were performed. The detail research contents and results of this paper could be summarized to five aspects as follow:1.Structure analysis and mechanism prediction of some energy transfer proteins in A.ferrooxidans.Cytochrome c:There are 10 cytochrome c proteins in A. ferrooxidans.Cluster analysis showed that they were classified to 4 homologous protein classes.The sequence alignment and structure compare for the 4 proteins of cytochrome c₄ family among them showed that the differences in the sites similar to Tyr63 and Gln121 in Cycl were responsible for their function differences.Cytochrome bc₁ complex:There are 2 cytochrome bc₁ complexes in A.ferrooxidans.Structure modeling and mechanism analysis were performed to them.The alignment and structure compare for them showed that the differences in the Arg79 site near to heme b_L in cytochrome b subunit for them were responsible for their differences of foreword or inverse function.Terminal oxidase:There are 4 terminal oxidases in A.ferrooxidans. Structure modeling and mechanism analysis were performed to them, results showed that the electron donors for cytochrome aa₃ and ba₃ complexes were cytochrome c proteins in periplasm,the electron donors for cytochrome bo₃ and bd complexes were quinones.Cytochrome CycA1:Modeled structure showed that this protein contained 2 hemes.Cytochrome Cyc2_iike protein:The bioinformatics analysis showed that this protein was a cytochrome c protein with one heme in outer membrane.Sulfide quinone reductase（SQR）:The structure of SQR is unresolved.The structure of SQR from A.ferrooxidans was modeled and docked with FAD and qunione,its catalyze mechanism was proposed based on these results.Gene doxDA:The bioinformatics analysis to doxDA-1 and doxDA-2 genes showed that they were both fused genes.DsbG protein:Modeled structure show that the two conserved residues Cys119 and Cys122 were very near but the place of them were far from the contact interface between two homo subunit.Ribose-5-phosphate isomerase A（RpiA）:Its structure was modeled and docked with R5P.Its key residues were identified and its catalyze mechanism was proposed based on these results.Glutathione reductase:Its structure was modeled and docked with substrates.Its key residues were identified and its catalyze mechanism was proposed based on these results.2.Structure modeling and protein expression verification of some energy transfer proteins in A.ferrooxidans.Rusticyanin:Electrostatic potential analysis,quantum chemistry computation for active site and protein expression and mutation confirmed that Cys138 was crucial for Cu atom binding. Iron oxidase（Iro）:The structure of Iro is unresolved.The structure of Iro from A.ferrooxidans was modeled,which showed that this protein was a high potential iron sulfur protein（HiPIP） family protein with a [Fe₄S₄]cluster coordinated by 4 cysteines.The virtual mutation experiment for Tyr10 predicted that Tyr10 was crucial for the stable of the[Fe₄S₄]cluster.Protein expression and mutation experiment further confirmed those conclusions.High potential iron sulfur protein（HiPIP）:Modeled structure show that this protein had a[Fe₄S₄]cluster coordinated by 4 cysteines. Protein expression showed that this protein had a[Fe₄S₄]cluster. Mutation experiment further confirmed the predicted residues coordinated[Fe₄S₄]cluster.Sulfite reductase:Structures of its flavin protein（SiR-FP） and heme protein（SiR-HP） are modeled.Results showed that SiR-HP had a[Fe₄S₄] cluster in the center coordinated by 4 cysteines,a heme is near to it. Protein expression showed that SiR-HP had a[Fe₄S₄]cluster and heme group.Mutation experiment further confirmed the predicted residues coordinate[Fe₄S₄]cluster.APS reductase:Modeled structure showed that this protein had a [Fe₄S₄]cluster coordinated by Cys110,Cys111,Cys193 and Cys196. Docking results showed that substrate APS was near to it.Protein expression confirmed that this protein had a[Fe₄S₄]cluster.Iron Sulfur Cluster Assembly sulfur donor protein（IscS）:Its structure was modeled and docked with cofactor pyridoxal 5’-phosphate （PLP） and substrate cysteine.Its key residues were identified.Protein expression confirmed that IscS was a cysteine desulfurase,which is a PLP-dependent enzyme that catalyzes the conversion of L-cysteine to L-alanine and sulfan sulfur to provide sulfur for iron sulfur cluster assembly.Iron Sulfur Cluster Assembly A protein（IseA）:IscA from A. ferrooxidans may contain[Fe₄S₄]or[Fe₂S₂]cluster.Modeled structure and protein mutation experiment confirmed that this protein had a[Fe₄S₄] cluster coordinated by Cys35,Cys99,Cys101 and Glu103.Iron Sulfur Cluster Assembly scaffold protein（IscU）:IscU from A. ferrooxidans may contain[Fe₄S₄]or[Fe₂S₂]cluster.Modeled structure and protein mutation experiment confirmed that this protein had a[Fe₂S₂] cluster coordinated by Cys37,Cys63,Cys106 and Asp39.Ferredoxin:Ferredoxin from A.ferrooxidans may contain[Fe₄S₄], [Fe₃S₄]or[Fe₂S₂]cluster.Modeled structure,protein expression and mutation experiment confirmed that this protein had a[Fe₂S₂]cluster coordinated by Cys42,Cys48,Cys51 and Cys87.Superoxide dismutase:Structure modeling and evolutionary trace analysis were performed to them,results show that the active center of it was an iron coordinated by Cys35,Cys99,Cys101 and Glu103.protein expression experiment further confirmed that this protein was an iron-containing superoxide dismutase.Medium-chain acyl-CoA dehydrogenase:Comparison for this protein and its Y375K mutation docked with acyl-CoA respectively and protein expressions and mutation experiments confirmed that mutation of Tyr375 to Lys375 allowed this protein to acquire acyl-CoA oxidase activity.Acyl-CoA oxidase:Molecular docking and protein expression experiment confirmed that Oct-2-en-4-ynoyl-CoA was a specific inhibitor of acyl-CoA oxidase.3.Molecular diversities of some multi-copies energy transfer proteins in A.ferrooxidans.In the level of three dimension molecular structure,molecular diversities of cytochrome c₄ proteins,rusticyanin,high potential iron sulfur protein and sulfide quinone reductase in all kinds of strains and their difference copies in same strain of A.ferrooxidans were studied. Results showed that the differences among multi-copies proteins in same strain were in the local of molecules;except for some minority strains,the difference sites of the same protein in difference strains were generally only several residues far from active centers.4.The energy transfer network map of iron and sulfur metabolism in the process of A.ferrooxidans leaching minerals.Systemically analyzing more than two handreds genes and more than thirty operons involving energy transfer in A.ferrooxidans,combining the above research results and the results of other people’s researches in literatures,the energy transfer pathway network map of iron and sulfur metabolism in the process of A.ferrooxidans leaching minerals was reconstructed.This map included mineral decompose,reaction in solution, and the subsystems of ferrous oxidation,sulfur metabolize,CO₂ fixation, N₂ fixation and H2 utilization of A.ferrooxidans.The sum of multicopies and isoenzymes of each protein and the redox potential of some key reactions were marked.Through this map,it can be known how an A. ferrooxidans to leach minerals.5.Analysis of the system of sulfide mineral bioleaehing and its acid produce coupled by energy transfer.With the new method of considering the flux of producing new matter as a cutting-point,the system of sulfide mineral bioleaching was systematically analyzed,the changes of this system were dominant by seven key matter flows of iron flow,sulfur flow,carbon flow,nitrogen flow,oxygen flow and metals flow.Finally,the details of acid produce coupled by energy transfer in the process of sulfide mineral bioleaching was analyzed.更多还原