Expression of human ferredoxin in Saccharomyces cerevisiae: mitochondrial import of the protein and assembly of the [2Fe-2S] center.
Vertebrate ferredoxins function in the transfer of reducing equivalents from NADPH:ferredoxin oxidoreductase to cytochrome P450 enzymes involved in steroid metabolism. We report here the expression of human mitochondrial ferredoxin in the yeast Saccharomyces cerevisiae. The full-length ferredoxin protein containing the ferredoxin mitochondrial leader sequence could not be stably expressed in S. cerevisiae, but a fusion protein consisting of the mature portion of ferredoxin linked to the mitochondrial leader sequence of the S. cerevisiae cytochrome c oxidase subunit Va protein (COX5a) could be stably expressed. The COX5a:ferredoxin fusion protein was targeted to the mitochondria as a preprotein and was cleaved at the normal processing site of the COX5a presequence during import into the matrix. Absorption spectra and electron transfer activity of the isolated fusion protein established that the [2Fe-2S] center was correctly assembled and incorporated into the recombinant ferredoxin in this heterologous system.
Seaton
B L
BL
Department of Physiology and Biophysics, University of California, Irvine 92717.
Vickery
L E
LE
D000595:Amino Acid Sequence; D003576:Electron Transport Complex IV / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D005288:Ferredoxins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D005800:Genes, Fungal; D006801:Humans; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011401:Promoter Regions, Genetic; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D013053:Spectrophotometry
D005288:Ferredoxins; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D003576:Electron Transport Complex IV