Contribution of a salt bridge to the thermostability of adrenodoxin determined by site-directed mutagenesis.
We identified a unique conserved salt bridge Arg89-Glu74 inside the protein core of adrenodoxin, which ensures proper orientation between the [2Fe-2S] cluster-containing domain and the recognition helix. Incorporation and geometry of the redox center were essentially preserved in the mutants E74D, R89A, and R89K as judged by EPR spectroscopy. However, absorption and CD spectra pointed out essential conformational changes in the protein vicinity of the [2Fe-2S] cluster. Judged by essentially increased K(m) and K(d) values and changed redox properties, mutations resulted in displacement of the recognition helix and hindered proper docking of the protein with both adrenodoxin reductase and CYP11A1. Substitutions of Arg89 and Glu74 induce thermodynamic destabilization attested by dramatically decreased unfolding temperature (T(d)) and enthalpy (Delta(d)H(T(d))). The heat capacity change of denaturation (Delta(d)C(p)) was significantly decreased for the mutants, suggesting that parts of the polypeptide chain normally hidden inside the protein core are exposed to the solvent in these variants.
Grinberg
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AV
Fachrichtung 8.8-Biochemie, Universität des Saarlandes, Saarbrücken, D-66041, Germany.
Bernhardt
R
R
D000325:Adrenodoxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D002786:Cholesterol Side-Chain Cleavage Enzyme / Q000378:metabolism; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D006358:Hot Temperature; D008958:Models, Molecular; D008962:Models, Theoretical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013053:Spectrophotometry
D000325:Adrenodoxin; D002786:Cholesterol Side-Chain Cleavage Enzyme; D005287:Ferredoxin-NADP Reductase