Effect of methionine80 heme coordination on domain swapping of cytochrome c
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Cytochrome c (cyt c) forms oligomers by domain swapping. It exchanges the C-terminal α-helical region between protomers, and the Met80‒heme iron bond is perturbed significantly in domain-swapped oligomers. The peroxidase activity of cyt c increases by Met80 dissociation from the heme iron, which may trigger apoptosis. This study elucidates the effect of the Met80 heme coordination on cyt c domain swapping by obtaining oligomers for both wild-type (WT) and M80A human cyt c by an addition of ethanol to their monomers, followed by lyophilization and dissolution to buffer, and investigating their dimer properties. The absorption and circular dichroism spectra of WT and M80A cyt c exhibited similar changes upon dimerization, indicating that Met80 does not affect the oligomerization process significantly. According to differential scanning calorimetric measurements, Met80 coordination to the heme iron had an effect on the stabilization of the monomer (ΔH = 16 kcal/mol), whereas no large difference was observed between the dimer-to-monomer dissociation temperatures of WT and M80A cyt c (61.0 °C). The activation enthalpy values were similar and relatively large for the dissociation of both WT and M80A cyt c dimers (WT, 120 ± 10 kcal/mol; M80A, 110 ± 10 kcal/mol), indicating that the dimers suffered large structural changes upon dissociation to monomers independent of the Met80 coordination to the heme iron. These results indicate that cyt c domain swapping may occur regardless of the Met80 coordination, whereas the monomer is stabilized by Met80 but the domain-swapped dimer structure and stability are less affected by the Met80 coordination.
KeywordsCytochrome c Domain swapping Methionine coordination Oligomerization
Differential scanning calorimetry
Fast protein liquid chromatography
Size exclusion chromatography
We thank Mr. Leigh McDowell, Nara Institute of Science and Technology, for his advice on manuscript preparation. This work was partially supported by Grants-in-Aid for Scientific Research from JSPS (Category B, No. JP26288080, S.H.; Challenging Exploratory Research, No. JP15K13744, S.H.; Scientific Research on Innovative Areas, No. JP16H00839, S.H.; Young Scientists B, No. JP16K17935, S.N.), Natural Science Foundation of Sichuan Province of China (No. 11ZB029, Z.W.), Open Project of Chemical Synthesis and Pollution Control Key Laboratory of Sichuan Province (No. CSPC2011-7-2, Z.W.), and Doctoral Fund of China West Normal University (No. 07B011, Z.W.).