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Mutations of tyrosine 82 in bovine adrenodoxin that affect binding to cytochromes P45011A1 and P45011B1 but not electron transfer

Item Type:Article
Title:Mutations of tyrosine 82 in bovine adrenodoxin that affect binding to cytochromes P45011A1 and P45011B1 but not electron transfer
Creators Name:Beckert, V. and Dettmer, R. and Bernhardt, R.
Abstract:To understand the function of the unique tyrosine in position 82 of bovine adrenodoxin (Adx), which had been proposed to be involved in electron transfer from NADPH-dependent adrenodoxin reductase (AdR) to cytochrome P-450 enzymes and/or AdR binding by chemical modification studies (Taniguchi, T., and Kimura, T. (1975) Biochemistry 14, 5573-5578), the residue was replaced by phenylalanine, leucine, or serine. Unchanged absorption, CD, and electron spin resonance spectra as well as redox potentials indicate that the environment of the [2Fe-2S] cluster was not affected by the mutations. The Vmax values in cytochrome c reduction, P45011A1- and P45011B1-dependent activities were also not changed when using Y82F, Y82S, and Y82L Adx mutants as electron donor, demonstrating that tyrosine 82 is not involved in the intra- or intermolecular electron transfer. Replacement of tyrosine 82 did not affect AdR binding as shown by unchanged cytochrome c activity. There are, however, changes in Km values up to 4-fold when measuring the enzymatic activities of mutant Adx with P45011A1 and P45011B1. These changes differ in dependence on the P-450 (P45011A1 or P45011B1) used. The results suggest that mutation of tyrosine 82 either directly or indirectly (by inducing small conformational changes of the binding domain) affects the binding of cytochromes P-450.
Keywords:Adrenal Cortex, Adrenodoxin, Amino Acid Sequence, Base Sequence, Binding Sites, Circular Dichroism, Cytochrome P-450 CYP2B1, Cytochrome P-450 Enzyme System, DNA Primers, Electron Transport, Kinetics, Mitochondria, Molecular Sequence Data, Oxidation-Reduction, Oxidoreductases, Polymerase Chain Reaction, Protein Binding, Protein Conformation, Recombinant Proteins, Site-Directed Mutagenesis, Tyrosine, Animals, Cattle
Source:Journal of Biological Chemistry
ISSN:0021-9258
Publisher:American Society for Biochemistry and Molecular Biology (U.S.A.)
Volume:269
Number:4
Page Range:2568-2673
Date:28 January 1994
Official Publication:http://www.jbc.org/content/269/4/2568.abstract
PubMed:View item in PubMed

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