Inversion of the stereochemistry around the sulfur atom of the axial methionine side chain through alteration of amino acid side chain packing in Hydrogenobacter thermophilus cytochrome c ₅₅₂ and its functional consequences

In cytochrome c, the coordination of the axial Met S_δ atom to the heme Fe atom occurs in one of two distinctly different stereochemical manners, i.e., R and S configurations, depending upon which of the two lone pairs of the S_δ atom is involved in the bond; hence, the Fe-coordinated S_δ atom becomes a chiral center. In this study, we demonstrated that an alteration of amino acid side chain packing induced by the mutation of a single amino acid residue, i.e., the A73V mutation, in Hydrogenobacter thermophilus cytochrome c₅₅₂ (HT) forces the inversion of the stereochemistry around the S_δ atom from the R configuration [Travaglini-Allocatelli, C., et al. (2005) J. Biol. Chem. 280, 25729-25734] to the S configuration. Functional comparison between the wild-type HT and the A73V mutant possessing the R and S configurations as to the stereochemistry around the S_δ atom, respectively, demonstrated that the redox potential (E_m) of the mutant at pH 6.00 and 25 C exhibited a positive shift of ∼20 mV relative to that of the wild-type HT, i.e., 245 mV, in an entropic manner. Because these two proteins have similar enthalpically stabilizing interactions, the difference in the entropic contribution to the E_m value between them is likely to be due to the effect of the conformational alteration of the axial Met side chain associated with the inversion of the stereochemistry around the S_δ atom due to the effect of mutation on the internal mobility of the loop bearing the axial Met. Thus, the present study demonstrated that the internal mobility of the loop bearing the axial Met, relevant to entropic control of the redox function of the protein, is affected quite sensitively by the contextual stereochemical packing of amino acid side chains in the proximity of the axial Met.