Versatility of non-native forms of human cytochrome c: pH and micellar concentration dependence.
In addition to its electron transfer activity, cytochrome c is now known to trigger apoptosis via peroxidase activity. This new function is related to a structural modification of the cytochrome upon association with anionic lipids, particularly cardiolipin present in the mitochondrial membrane. However, the exact nature of the non-native state induced by this interaction remains an active subject of debate. In this work, using human cytochromes c (native and two single-histidine mutants and the corresponding double mutant) and micelles as a hydrophobic medium, we succeeded, through UV-visible spectroscopy, circular dichroism spectroscopy and NMR spectroscopy, in fully characterizing the nature of the sixth ligand replacing the native methionine. Furthermore, careful pH titrations permitted the identification of the amino acids involved in the iron binding over a range of pH values. Replacement of the methionine by lysine was only observed at pH above 8.5, whereas histidine binding is dependent on both pH and micelle concentration. The pH variation range for histidine protonation is relatively narrow and is consistent with the mitochondrial intermembrane pH changes occurring during apoptosis. These results allow us to rule out lysine as the sixth ligand at pH values close to neutrality and reinforce the role of histidines (preferentially His33 vs. His26) as the main candidate to replace methionine in the non-native cytochrome c. Finally, on the basis of these results and molecular dynamics simulations, we propose a 3D model for non-native cytochrome c in a micellar environment.
Université de Rennes 1 and UMR CNRS 6290, SIM, PRISM, Biosit, CS 34317, Campus de Villejean, 35043 Rennes Cedex, France.
Matthieu
M
Simon
Valérie
V
Metzinger-Le Meuth
Soizic
S
Chevance
Olivier
O
Delalande
Arnaud
A
Bondon
D045304:Cytochromes c / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008823:Micelles; D056004:Molecular Dynamics Simulation; D009154:Mutation; D010743:Phospholipids / Q000378:metabolism; D011487:Protein Conformation; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
D008024:Ligands; D008823:Micelles; D010743:Phospholipids; D012967:Sodium Dodecyl Sulfate; D006418:Heme; D045304:Cytochromes c