Coexistence of Native-like and Non-Native Partially
Unfolded Ferricytochrome <i>c</i> on the Surface of Cardiolipin-Containing
Liposomes
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Abstract
Cytochrome <i>c</i>, in spite of adopting a rather rigid
structure around its prosthetic heme group, is rather diverse with
regard to its function and structural variability. On the surface
of the inner membrane of mitochondria it serves as an electron transfer
carrier. However, at conditions which have not yet been unambiguously
identified, cytochrome <i>c</i> can adopt a variety of non-native
conformations, some of which exhibit peroxidase activity. Cardiolipin-containing
liposomes have served as ideal model system to investigate the various
modes of interaction between cytochrome <i>c</i> and the
inner mitochrondrial membrane. We probed the binding of horse heart
ferricytochrome <i>c</i> to liposomes formed with 20% tetraoleoyl
cardiolipin (TOCL) and 80% dioleoyl-<i>sn</i>-glycero-3-phosphocholine
(DOPC) as a function of lipid/protein ratio by fluorescence and visible
circular dichroism spectroscopy. The obtained binding isotherms suggest
that they reflect reversible binding processes, which excludes the
possibility of significant protein insertion into the membrane. A
global analysis of our data revealed the existence of two binding
sites on the protein which causes rather different degrees of protein
unfolding. We found that these two modes of interaction between protein
and liposome led to conformational changes. While site 1 is relatively
unaffected by NaCl, site 2 shows a more native-like state or a higher
population of the native state in the presence of NaCl. At the highest
utilized concentration of NaCl, there is only a 40% inhibition of
the binding to site 2. We interpret our finding for this binding site
as reflecting an equilibrium between electrostatically bound proteins
with a high degree of unfolding and less unfolded proteins which bind
either via H-bonding between lysine side chains and PO<sub>2</sub><sup>β</sup> or hydrophobic interactions. With regard to site
2 binding, our results are reminiscent of the two-state equilibrium
between a compact C and an extended E-state proposed by Pletneva and
co-workers (Hanske et al. <i>Proc. Natl. Acad. Sci. U.S.A.</i> <b>2012</b>, <i>109</i>, 125β230). We conjecture
that the nonelectrostatically bound proteins should have higher abilities
to maintain the redox potential that is required for the function
as an electron transfer protein