Cytochrome c polymerization by successive domain swapping at the C-terminal helix

Abstract

Cytochrome c (cyt c ) is a stable protein that functions in a monomeric state as an electron donor for cytochrome c oxidase. It is also released to the cytosol when permeabilization of the mitochondrial outer membrane occurs at the early stage of apoptosis. For nearly half a century, it has been known that cyt c forms polymers, but the polymerization mechanism remains unknown. We found that cyt c forms polymers by successive domain swapping, where the C-terminal helix is displaced from its original position in the monomer and Met-heme coordination is perturbed significantly. In the crystal structures of dimeric and trimeric cyt c , the C-terminal helices are replaced by the corresponding domain of other cyt c molecules and Met80 is dissociated from the heme. The solution structures of dimeric, trimeric, and tetrameric cyt c were linear based on small-angle X-ray scattering measurements, where the trimeric linear structure shifted toward the cyclic structure by addition of PEG and (NH 4 ) 2 HPO 4 . The absorption and CD spectra of high-order oligomers (∼40 mer) were similar to those of dimeric and trimeric cyt c but different from those of monomeric cyt c . For dimeric, trimeric, and tetrameric cyt c , the Δ H of the oligomer dissociation to monomers was estimated to be about -20 kcal/mol per protomer unit, where Met-heme coordination appears to contribute largely to Δ H . The present results suggest that cyt c polymerization occurs by successive domain swapping, which may be a common mechanism of protein polymerization.