Influence of cysteine-directed mutations at the Ω-loops on peroxidase activity of human cytochrome c

Abstract

Cytochrome c (Cytc) is a multifunctional protein associated with electron shuttling in the inner membrane of mitochondria and also involving in the apoptotic pathway. It has been identified that mutations located in the flexible central 40-57 Ω-loop including the naturally occurring G41S, Y48H, and A51V mutants, which are found in patients with thrombocytopenia 4, a platelet disorder, alter the structural properties of human Cytc (hCytc) that associated to enhanced peroxidase activity. In this work we compared the cysteine-directed mutants of hCytc located in three different parts of Ω-loops, i.e., T28C and G34C (proximal Ω-loop), and A50C (central Ω-loop), with respect to the wild-type (WT) hCytc. The mutants and WT hCytc were structurally characterized by circular dichroism, heating and chemical denaturations, and fluorescence spectroscopy. The flexibility at the cysteine mutated sites was directly determined by site-directed spin-labeling Electron Spin Resonance. Alkaline transitions were determined by pH titration and the alkaline conformers were related to peroxidase activity of all hCytc proteins. Structural and dynamic characterizations were rationally correlated to the modulation of peroxidase activity in these mutants in comparison to the WT hCytc. We found that the cysteine mutations at residues T28 and G34, both located in the same region of Ω-loop, developed different conformations and dynamical properties that lead to different effects on the rates of peroxidase activity (G34C was ~2.6 folds higher), whereas the rate of G34C was closer to that of A50C mutant. The results implied that the flexibility and local structures of the proximal Ω-loop could also play an important role in modulating the peroxidase activity which can be associated to apoptosis.