4 research outputs found
Coexistence of Native-like and Non-Native Partially Unfolded Ferricytochrome <i>c</i> on the Surface of Cardiolipin-Containing Liposomes
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>–</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>, 125–230). 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
Coexistence of Native-Like and Non-Native Cytochrome <i>c</i> on Anionic Liposomes with Different Cardiolipin Content
We employed a combination of fluorescence,
visible circular dichroism,
and absorption spectroscopy to study the conformational changes of
ferricytochrome <i>c</i> upon its binding to cardiolipin-containing
small unilamellar vesicles. The measurements were performed as a function
of the cardiolipin concentration, the cardiolipin content of the liposomes,
and the NaCl concentration of the solvent. The data were analyzed
with a novel model that combines a single binding step with a conformational
equilibrium between native-like and non-native-like proteins bound
to the membrane surface. The equilibrium between the two conformations,
which themselves are comprised of structurally slightly different
subconformations, shifts to the more non-native-like conformation
with increasing cardiolipin concentration. For the binding isotherms
described in this paper, we explicitly considered the enthalpic and
entropic contributions of molecular crowding to protein binding at
low lipid concentrations and high occupancy of the liposome surface.
Increasing the CL content of liposomes increases the overall binding
affinity but makes the conformational distribution much more susceptible
to the influence of sodium and chloride ions, which shifts the equilibrium
toward the more native-like state and directly inhibits binding, particularly
to liposomes with 100% cardiolipin content. Spectroscopic evidence
further suggests that a fraction of the non-native conformers adopts
a pentacoordinated state similar to those obtained in class C peroxidases.
On the basis of our results, we propose a hypothesis that describes
the balance between facilitating and impeding forces controlling the
peroxidase activity of cytochrome <i>c</i> in the inner
membrane space of mitochondria
The (Not Completely Irreversible) Population of a Misfolded State of Cytochrome <i>c</i> under Folding Conditions
This paper reports the discovery of a (meta)Âstable partially
unfolded
state of horse heart ferricytochrome <i>c</i> that was obtained
after exposing the protein to a solution with an alkaline pH of 11.5
for 1 week. Thereafter, the protein did not undergo any detectable
change in its secondary and tertiary structure upon adjusting the
solution to folding promoting conditions at neutral pH. Spectroscopic
data suggest that the misfolded protein exhibits a hexacoordinated
low-spin state with a hydroxyl ion as the likely ligand. Below pH
6, a new ligation state emerges with the spectroscopic characteristics
of a pentacoordinated quantum mixed state of the heme iron. Gel electrophoresis
revealed substantial formation of soluble dimers and trimers at submillimolar
concentrations, whereas monomers were dominant at lower, micromolar
concentrations. Ultraviolet circular dichroism spectra indicate that
oxidized monomers are pre-molten globule to globule-like with a substantial
fraction of secondary (helical) structure reminiscent of alkaline
state V. The oligomers contain even more helical structure, which
suggests domain swapping as the underlying mechanism of their formation.
A substantial fraction of the submillimolar mixture of monomers and
oligomers underwent a reduction of the heme iron. Its dependence on
pH suggests the coupling to a proton transfer process. Altogether,
our data indicate a partially unfolded ferricytochrome <i>c</i> conformation with spectroscopic characteristics reminiscent of the
recently discovered alkaline isomer V<sub>b</sub>, which is stabilized
under folding conditions by exposing the protein to a very alkaline
pH for an extended period of time
Autoxidation of Reduced Horse Heart Cytochrome <i>c</i> Catalyzed by Cardiolipin-Containing Membranes
Visible circular
dichroism, absorption, and fluorescence spectroscopy
were used to probe the binding of horse heart ferrocytochrome <i>c</i> to anionic cardiolipin (CL) head groups on the surface
of 1,1′,2,2′-tetraoleoyl cardiolipin (TOCL)/1,2-dioleoyl-<i>sn</i>-glycero-3-phosphocholine (DOPC) (20%:80%) liposomes in
an aerobic environment. We found that ferrocytochrome <i>c</i> undergoes a conformational transition upon binding that leads to
complete oxidation of the protein at intermediate and high CL concentrations.
At low lipid concentrations, the protein maintains a structure that
is only slightly different from its native one, whereas an ensemble
of misligated predominantly hexacoordinated low-spin states become
increasingly populated at high lipid concentrations. A minor fraction
of conformations with either high- or quantum-mixed-spin states were
detected at a CL to protein ratio of 200 (the largest one investigated).
The population of the non-native state is less pronounced than that
found for cytochrome <i>c</i>–CL interactions initiated
with oxidized cytochrome <i>c</i>. Under anaerobic conditions,
the protein maintains its reduced state but still undergoes some conformational
change upon binding to CL head groups on the liposome surface. Our
data suggest that CL-containing liposomes function as catalysts by
reducing the activation barrier for a Fe<sup>2+</sup> → O<sub>2</sub> electron transfer. Adding NaCl to the existing cytochrome–liposome
mixtures under aerobic conditions inhibits protein autoxidation of
ferrocytochrome <i>c</i> and stabilizes the reduced state
of the membrane-bound protein