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 geo metry of the redox center were essentially preserved in the mutants E74D, R 89A, and R89K as judged by EPR spectroscopy. However, absorption and CD spe ctra pointed out essential conformational changes in the protein vicinity o f the [2Fe-2S] cluster. Judged by essentially increased K-m and K-d values and changed redox properties, mutations resulted in displacement of the rec ognition helix and hindered proper docking of the protein with both adrenod oxin reductase and CYP11A1 Substitutions of Arg89 and Glu74 induce thermody namic destabilization attested by dramatically decreased unfolding temperat ure (T-d) and enthalpy (Delta H-d(T-d)). The heat capacity change of denatu ration (Delta C-d(p)) was significantly decreased for the mutants, suggesti ng that parts of the polypeptide chain normally hidden inside the protein c ore are exposed to the solvent in these variants. (C) 2001 Elsevier Science .