C-terminal region of adrenodoxin affects its structural integrity and determines differences in its electron transfer function to cytochrome P-450.
The role of the C-terminal region of adrenodoxin was studied by analyzing deletion mutants 4-114 and 4-108 lacking amino acids 1-3 and 115-128 or 109-128, respectively. Absorption spectra of these mutants were found to be identical to that of wild type adrenodoxin. However, EPR and CD studies indicated that the structure of deletion mutants 4-114 and 4-108 differs from that of wild type adrenodoxin. Mutant 4-107, which in addition to residues 109-128 lacks the unique proline 108, showed no EPR spectrum. This indicates that proline 108 plays an essential role for the formation of the iron-sulfur cluster. Deletion of residues 115-128 or 109-128 did not essentially affect adrenodoxin reductase binding as shown by nearly unchanged cytochrome c reduction activity. In a CYP11A1 assay, mutants 4-108 and 4-114 exhibited 3.2- and 5-fold decreased Km values, respectively, whilst the Kd values for CYP11A1 decreased 3- and 1.9-fold, respectively. Additionally, in a CYP11B1 assay, mutants 4-108 and 4-114 showed decreased Km values. Furthermore, the first step of electron transfer to CYP11B1, but not to CYP11A1, was accelerated up to 4.5-fold by the adrenodoxin mutants. The results suggest that the C-terminal peptide of adrenodoxin, especially proline 108, affects the structural integrity of the iron-sulfur cluster and that electron donation from adrenodoxin to CYP11A1 and CYP11B1 is determined at least in part by different features of the cytochromes.
Uhlmann
H
H
Max-Delbrück-Centrum für Molekulare Medizin, Berlin, Federal Republic of Germany.
Kraft
R
R
Bernhardt
R
R
D000311:Adrenal Glands / Q000201:enzymology; D000325:Adrenodoxin / Q000096:biosynthesis / Q000737:chemistry* / Q000494:pharmacology*; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D017931:DNA Primers; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport / Q000187:drug effects; D004926:Escherichia coli; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000494:pharmacology; D017384:Sequence Deletion
D017931:DNA Primers; D011994:Recombinant Proteins; D000325:Adrenodoxin; D003577:Cytochrome P-450 Enzyme System; D005287:Ferredoxin-NADP Reductase