Investigating peroxidase activity and cardiolipin oxidation in human cytochrome c variants

Cytochrome c is a multifunctional heme protein with roles in apoptosis as well as mitochondrial electron transport. Naturally occurring mutations in cytochrome c cause autosomal dominant thrombocytopenia by an unknown mechanism. The peroxidase activity of cytochrome c is proposed to contribute to apoptosis by peroxidation of cardiolipin in the mitochondrial inner membrane. However, cytochrome c heme is hexa-coordinate with a methionine (Met80) on the distal side, stopping it from acting as an efficient peroxidase. The first naturally occurring variant of cytochrome c, G41S, has higher peroxidase activity than wild-type. To understand the basis for this increase and gain insights into the peroxidase activity of wild-type we have studied wild-type, G41S and the unnatural variant G41T. Using a combined kinetic and mass spectrometric analysis, we show that H 2 O 2 specifically oxidizes methionine 80 to the sulfoxide. In the absence of substrate this is oxidized to the sulfone, decreasing peroxidase activity. In the presence of substrate, the sulfoxide is the main oxidation product. Peroxidase activity correlates with the proportion of sulfoxide present and when fully in that form all variants have the same activity. The implications for cardiolipin oxidation are now being investigated.