A comprehensive guide through the fold and stability of cytochrome c with implications for disease

Cytochrome c (Cyt-c), encoded by CYCS gene, is crucial for electron transport, peroxidase activity, and apoptosis. Mutations in CYCS can cause thrombocytopenia 4 (THC4), a disorder with low platelet counts. We have, for instance, recently described six Italian families with five different heterozygous missense CYCS variants. These mutations likely enhance peroxidase and apoptotic activities, yet mechanisms causing reduced platelet production and increased apoptosis are unclear. This study investigates all clinically important Cyt-c variants using an integrated bioinformatics approach. Our findings reveal that all variants are at evolutionarily conserved sites, likely disrupting Cyt-c function and contributing to disease phenotypes. Specific variants likely affect phosphorylation (T20I, V21G, Y49H), ubiquitination (G42S, A52T, A52V), and glycosylation (H27Y, N32H, L33V, G42S). Cyt-c variants may influence the function of interacting genes and proteins involved in apoptosis and mitochondrial function. Molecular dynamics simulations reveal significant structural differences from the wild-type, with mutants showing reduced stability and increased flexibility, particularly in the omega loops. These changes weaken hydrogen bonds and increase distances between key residues, opening the heme active site. This open conformation likely enhances accessibility to small molecules such as H₂O₂, thereby promoting peroxidase activity, which may enhance apoptosis and likely impact megakaryopoiesis and platelet homeostasis in THC4.