Identification of free and [Fe2S2]-bound cysteine residues of adrenodoxin.
Bovine adrenodoxin was labeled with 5-iodoacetamidofluorescein to determine which of the five cysteines is free and which participate in iron coordination. Native protein was labeled at two stoichiometries, 0.15:1 and 1:1, both of which produced a single fluorescent product. Labeled tryptic peptides were isolated from both preparations and identified as residues 90-98 with 5-acetamidofluorescein cysteine at residue 95. From the preparation labeled at 0.15:1 stoichiometry, the fraction of tryptic peptide containing nonlabeled cysteines 92 and 95 was isolated and identified; this peptide was shown to be absent in the sample labeled at 1:1 stoichiometry. 5-Acetamidofluorescein-labeled adrenodoxin supported electron transport with adrenodoxin reductase and cytochromes P-450sec and P-45011 beta, demonstrating that labeling occurred without disruption of the iron-sulfur center. These results identify cysteine 95 as the most reactive and single free thiol in native adrenodoxin and imply the role of cysteine residues 46 [corrected], 52, 55, and 92 in iron-sulfur coordination.
Cupp
J R
JR
Department of Physiology and Biophysics, University of California, Irvine 92717.
Vickery
L E
LE
D000302:Adrenal Cortex / Q000378:metabolism; D000325:Adrenodoxin / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003545:Cysteine; D005452:Fluoresceins / Q000494:pharmacology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D011549:Pseudomonas / Q000378:metabolism; D013045:Species Specificity
D005452:Fluoresceins; D010446:Peptide Fragments; D000325:Adrenodoxin; C015296:5-iodoacetamidofluorescein; D003545:Cysteine