Expression and spectroscopic characterization of the hydrogenosomal [2Fe-2S] ferredoxin from the protozoan Trichomonas vaginalis.
The heterologous expression and spectroscopic characterization of the [2Fe-2S] ferredoxin from the sexually transmitted human parasite Trichomonas vaginalis is described. Using oligonucleotide primers based on the deduced DNA sequence, the gene encoding the ferredoxin was amplified by polymerase chain reaction and cloned into a T7 RNA polymerase expression vector. Expression of the gene in Escherichia coli host HMS174(DE3) resulted in the high level production of the protein with the correctly assembled iron-sulfur cluster. The absorption, circular dichroism, resonance Raman, and EPR spectra of the recombinant protein revealed many differences from those of other [2Fe-2S] ferredoxins. The redox potential of the protein (-347 mV versus normal hydrogen electrode) was also determined. Whereas the amino acid sequence of T. vaginalis ferredoxin showed greatest homology to the [2Fe-2S] ferredoxins found in bacteria and vertebrate mitochondria which function in cytochrome P450 oxidation pathways, the spectroscopic properties showed substantial dissimilarity. Differences in the biophysical properties and function of T. vaginalis ferredoxin are proposed to result from the characteristic amino acid sequence of the parasite protein near the cysteine residues that ligate the valence-localized Fe(III) site of the reduced cluster.
Vidakovic
M S
MS
Department of Chemistry, University of Houston, Houston, Texas 77204-5641, USA.
Fraczkiewicz
G
G
Germanas
J P
JP
D000325:Adrenodoxin / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001419:Bacteria / Q000378:metabolism; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli; D005288:Ferredoxins / Q000096:biosynthesis* / Q000737:chemistry*; D017125:Genes, Protozoan; D006801:Humans; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D013698:Templates, Genetic; D014246:Trichomonas vaginalis / Q000378:metabolism*; D014714:Vertebrates; D014764:Viral Proteins
D017931:DNA Primers; D005288:Ferredoxins; D011994:Recombinant Proteins; D014764:Viral Proteins; D000325:Adrenodoxin; C100137:putidaredoxin; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases