Study Of Iron-sulfur Cluster Assembly Protein IscU And Secreted Proteins From Acidithiobacillus Ferrooxidans

Acidithiobacillus ferrooxidans(A.ferrooxidans)is one of the most studied microorganisms commonly found in bioleaching and acid mine drainage.Understanding the respiration of iron and reduced sulfur compounds is of wide interest,not only scientifically,but also from an economic and environmental point of view.To gain further insight into the physiological role of the known redox proteins and to help identify yet unknown or missing components involved in iron and sulfur respiratory chains to oxygen,the iron-sulfur cluster assembly scaffold protein IscU and the secreted proteins encoded by genes in genome of A.ferrooxidans were studied.The gene of IscU was cloned and expressed in Escherichia coli.The protein was purified by one-step affinity chromatography to homogeneity. The purity of the IscU and its mutant proteins was further examined by SDS-PAGE and single bands corresponding to the 16 kDa.The protein was in apo-form.Incubation of apoIscU anaerobically with Fe²⁺and sulfide in the presence of dithiothreitol resulted in a brownish protein containing an iron-sulfur cluster.The UV-visible and EPR spectra of the reconstitute IscU showed the typical signal for proteins containing iron-sulfur clusters, 415nm and g value 2.013 respectively,indicating that[Fe₂S₂]cluster could be assembled in apoIscU with Fe²⁺and sulfide in vitro.The[Fe₂S₂] cluster was also successfully assembled in apo-IscU in vitro in the presence of two scaffold proteins of IscA and IscS,which utilize Fe²⁺and L-cysteine to synthesize the cluster.The absorption at 415 nm increased with time when L-cysteine was added to the reaction mixture,indicating the formation of[Fe₂S₂]cluster,and EPR spectra result also confirmed that the iron-sulfur cluster was assembled in the protein.Site-directed mutagenesis and the modeled overall structure for the protein revealed that Cys37,Asp39,Cys63 and Cys 106 were involved in ligating with the[Fe₂S₂]cluster.Determining the subcellular localization of a protein is an important first step toward understanding its function.The internet-based softwares SignalP v3.0,TMHMM v2.0,TatP v1.0,LipoP v1.0 and SecretomeP v2.0 were combined to predict the signal peptides from the 3137 ORFs in Acidithiobacills ferrooxidans ATCC 23270 genome.462 proteins were predicted to be secreted among 3137 proteins,among which 127 proteins have Sec-type signal peptides,13 proteins have twin-arginine signal peptides,38 proteins have lipoprotein signal peptides and 325 non-classical proteins.The majority(69.9%)of the 462 periplasmic proteins of this acidophilic microorganism showed pI values higher than 7. The length of the Sec-type signal peptides was different from 20 to 30 amino acids,and the most dominant one was 23 amino acids in length.The size of most ORFs varies between 100～500 amino acids.The frequency of particular amino acids in signal peptides by statistical approach was 68.08%for non-polar,31.92%for polar,3.42%for negative and 11.10% for positive amino acids.The most frequent amino acid in signal peptides was alanine,and the least was aspartate.The most frequent amino acid in the position -3,-1 and +1 was alanine,68.47%,89.19%and 31.53%, respectively.Among the top 100 scoring non-classical proteins,36 were provided with function description,mainly cell envelope,energy metabolism,transport and binding proteins.These results implied that processes catalyzed by these 462 proteins probably occurred in the periplasm or outside of cells,at least partially.