EXPRESSION AND SPECTROSCOPIC CHARACTERIZATION OF THE HYDROGENOSOMAL [2FE-2S] FERREDOXIN FROM THE PROTOZOAN TRICHOMONAS-VAGINALIS

heterologous expression and spectroscopic characterization of the [2Fe -2S] ferredoxin from the sexually transmitted human parasite Trichomon as vaginalis is described. Using oligonucleotide primers based on the deduced DNA sequence, the gene encoding the ferredoxin was amplified b y polymerase chain reaction and cloned into a T7 RNA polymerase expres sion vector. Expression of the gene in Escherichia coli host HMS174(DE 3) resulted in the high level production of the protein with the corre ctly 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) wa s also determined, Whereas the amino acid sequence of T. vaginalis fer redoxin 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 d issimilarity. Differences in the biophysical properties and function o f T. vaginalis ferredoxin are proposed to result from the characterist ic amino acid sequence of the parasite protein near the cysteine resid ues that ligate the valence-localized Fe(III) site of the reduced clus ter.