2 research outputs found
pH Dependence of Ferricytochrome <i>c</i> Conformational Transitions during Binding to Cardiolipin Membranes: Evidence for Histidine as the Distal Ligand at Neutral pH
The conformational
changes of ferricytochrome <i>c</i> upon binding to cardiolipin-containing
small unilamellar vesicles
were studied at slightly acidic pH using fluorescence, visible circular
dichroism, UV–visible absorption, and resonance Raman spectroscopy.
The obtained spectroscopic response data suggest a mode of interaction,
which is clearly distinct from the binding process observed at neutral
pH. Evidence of a reversible and electrostatic binding mechanism under
these conditions is provided through binding inhibition in the presence
of 150 mM NaCl. Moreover, UV–visible absorption and resonance
Raman spectra reveal that the conformational ensemble of membrane
bound cytochrome <i>c</i> is dominated by a mixture of conformers
with pentacoordinated and hexacoordinated high-spin heme irons, which
contrast with the dominance of low-spin species at neutral pH. While
our results confirm the L-site binding proposed by Kawai et al., they
point to the protonation of a histidine ligand (H33) as conformational
trigger
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