👤 Bruce E. Bowler

A 2.08 Å structure of an alkaline conformer of the domain-swapped dimer of K72A human cytochrome c (Cytc) crystallized at pH 9.9 is presented. In the structure, Lys79 is ligated to the heme. All other domain-swapped dimer structures of Cytc have water bound to this coordination site. Part of Ω-loop D (residues 70-85) forms a flexible linker between the subunits in other Cytc domain-swapped dimer structures but instead converts to a helix in the alkaline conformer of the dimer combining with the C-terminal helix to form two 26-residue helices that bracket both sides of the dimer. The alkaline transition of the K72A human dimer monitored at both 625 nm (high spin heme) and 695 nm (Met80 ligation) yields midpoint pH values of 6.6 and 7.6, respectively, showing that the Met80 → Lys79 and high spin to low spin transitions are distinct. The dimer peroxidase activity increases rapidly below pH 7, suggesting that population of the high spin form of the heme is what promotes peroxidase activity. Comparison of the structures of the alkaline dimer and the neutral pH dimer shows that the neutral pH conformer has a better electrostatic surface for binding to a cardiolipin-containing membrane and provides better access for small molecules to the heme iron. Given that the pH of mitochondrial cristae ranges from 6.9 to 7.2, the alkaline transition of the Cytc dimer could provide a conformational switch to tune the peroxidase activity of Cytc that oxygenates cardiolipin in the early stages of apoptosis. Show less

The A51V variant of human cytochrome c is linked to thrombocytopenia 4 (THC4), a condition that causes decreased blood platelet counts. A 1.82 Å structure of the A51V variant shows only minor changes in tertiary structure relative to the wild-type (WT) protein. Guanidine hydrochloride denaturation demonstrates that the global stability of the A51V variant is 1.3 kcal/mol less than that of the WT protein. The midpoint pH, pH_1/2, of the alkaline transition of the A51V variant is 1 unit less than that of the WT protein. Stopped-flow pH jump experiments show that the A51V substitution affects the triggering ionization for one of two kinetically distinguishable alkaline conformers and enhances the accessibility of a high-spin heme transient. The pH_1/2 for acid unfolding of the A51V variant is 0.7 units higher than for that of the WT protein. Consistent with the greater accessibility of non-native conformers for the A51V variant, the k_cat values for its peroxidase activity increase by 6- to 15-fold in the pH range of 5-8 versus those of the WT protein. These data along with previously reported data for the other THC4-linked variants, G41S and Y48H, underscore the role of Ω-loop C (residues 40-57) in modulating the peroxidase activity of cytochrome c early in apoptosis. Show less

Cytochrome c binds to cardiolipin (CL) on the inner mitochondrial membrane during the initial stages of apoptosis where it oxidizes CL, promoting its release into the cytoplasm where it initiates apoptosis. Previous work has identified interaction sites on cytochrome c involved in the cytochrome c-CL interaction. The contributions of the lysines attributed to site A, the anionic site, are studied here to elucidate the relative importance of each for electrostatic interaction of cytochrome c with CL at pH 8, conditions where site A is dominant. A set of single, double, and quadruple lysine to alanine variants of yeast iso-1-cytochrome c, at sequence positions 72, 73, 86, and 87, show that all contribute to the site A-mediated interaction with CL. All variants experience two sequential structural rearrangements as the lipid to protein ratio (LPR) increases. At a low LPR near 10, all variants undergo a small heme-centered structural change detected by Soret circular dichroism. At higher LPRs ranging from 22 to 34, all variants partially unfold as detected by Trp59 emission. The robustness of the mechanism of interaction to sequential neutralization of the four lysines assigned to site A demonstrates that site A is more extensive than previously supposed. The nature of both structural rearrangements also depends on which lysines constitute site A. The peroxidase activity of cytochrome c in the early stages of apoptosis depends on the nature of structural rearrangement near the heme. Thus, the lysines that comprise site A may have evolved to optimize the peroxidase signaling switch. Show less