The G41S Variant of Human Cytochrome C Enhances Apoptosis via Increased Dynamics

Abstract

In addition to its well-known role as a component of the electron transport chain, mitochondrial cytochrome c can also act as a peroxidase in the early stages of the intrinsic apoptosis pathway. Ferricytochrome c contains a hexacoordinated heme iron, for which H18 and M80 are the axial ligands. Residual peroxidase activity under native conditions arises from a minor populated pentacoordinated form, in which M80 is not coordinated to the heme iron. At the onset of apoptosis, binding to cardiolipin increases the proportion of the pentacoordinated form, increasing peroxidase activity which eventually leads to apoptosis. Mutations in the human gene for cytochrome c, which result in enhanced mitochondrial apoptotic activity, cause thrombocytopenia 4, an inherited autosomal dominant thrombocytopenia, characterised by a deficiency in the number of platelets in the blood and leading to abnormal bleeding. The first such mutation to be reported was G41S. Here we use stopped-flow kinetic studies of azide binding to human ferricytochrome c, backbone amide H/D exchange and 15N-relaxation dynamics measured by NMR spectroscopy to compare the wild type and G41S forms of human cytochrome c. We show that alternative conformations are kinetically and thermodynamically more readily accessible for the G41S variant than for the wild-type protein. Residue 41 is located in the 40-57 Ω-loop, and the increased loop dynamics in the G41S variant promote the dissociation from the heme iron of the M80 ligand, revealing a direct conformational link between the loop and the axial ligand to the heme iron. Increased dissociation of M80 increases the population of a peroxidase active state, which is a key non-native conformational state in apoptosis.