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C-terminal region of adrenodoxin affects its structural integrity and determines differences in its electron transfer function to cytochrome P-450

Item Type:Article
Title:C-terminal region of adrenodoxin affects its structural integrity and determines differences in its electron transfer function to cytochrome P-450
Creators Name:Uhlmann, H. and Kraft, R. and Bernhardt, R.
Abstract: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.
Keywords:Adrenal Glands, Adrenodoxin, Affinity Chromatography, Base Sequence, Circular Dichroism, Cytochrome P-450 Enzyme System, DNA Primers, Electron Spin Resonance Spectroscopy, Electron Transport, Escherichia Coli, Ferredoxin-NADP Reductase, Kinetics, Molecular Cloning, Molecular Sequence Data, Mutagenesis, Polymerase Chain Reaction, Recombinant Proteins, Sequence Deletion, Animals, Cattle
Source:Journal of Biological Chemistry
ISSN:0021-9258
Publisher:American Society for Biochemistry and Molecular Biology (U.S.A.)
Volume:269
Number:36
Page Range:22557-22564
Date:9 September 1994
Official Publication:http://www.jbc.org/content/269/36/22557.abstract
PubMed:View item in PubMed

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