Study On The System Of Iron And Sulfur Metabolizm Of The Process Of Acidithiobacillus Ferrooxidans Leaching Minerals

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.