Investigation of the role of a surface patch in the self-association of Chromatium vinosum high potential iron-sulfur protein

The role of a flattened, relatively hydrophobic surface patch in the self-a ssociation of Chromatium vinosum HiPIP was assessed by substituting phenyla lanine 48 with lysine. The reduction potential of the F48K variant was 26 m V higher than that of the wild-type (WT) recombinant (rc) HiPIP, consistent with the introduction of a positive charge close to the cluster. Nuclear m agnetic resonance spectroscopy (NMR) revealed that the electronic structure of the oxidized cluster in these two proteins is very similar at 295 K. In contrast, the electron transfer self-exchange rate constant of F48K was at least 15-fold lower than that of the WT rcHiPIP, indicating that the intro duction of a positive charge at position 48 diminishes self-association of the HiPIP in solution. Moreover, the substitution at position 48 abolished the fine structure in the g(z) region of the electron paramagnetic resonanc e (EPR) spectrum of oxidized C. vinosum rcHiPIP recorded in the presence of 1 M sodium chloride. These results support the hypothesis that the flatten ed, relatively hydrophobic patch mediates interaction between two molecules of HiPIP and that freezing-induced dimerization of the HiPIP mediated by t his patch is responsible for the unusual fine structure observed in the EPR spectrum of the oxidized C. vinosum HiPIP. (C) 1999 Elsevier Science B.V. All rights reserved.