6 research outputs found
Ligation and Reactivity of Methionine-Oxidized Cytochrome <i>c</i>
Met80, one of the
heme iron ligands in cytochrome <i>c</i> (cyt <i>c</i>), is readily oxidized to Met sulfoxide (Met-SO) by several biologically
relevant oxidants. The modification has been suggested to affect both
the electron-transfer (ET) and apoptotic functions of this metalloprotein.
The coordination of the heme iron in Met-oxidized cyt <i>c</i> (Met-SO cyt <i>c</i>) is critical for both of these functions
but has remained poorly defined. We present electronic absorption,
NMR, and EPR spectroscopic investigations as well as kinetic studies
and mutational analyses to identify the heme iron ligands in yeast <i>iso</i>-1 Met-SO cyt <i>c</i>. Similar to the alkaline
form of native cyt <i>c</i>, Lys73 and Lys79 ligate to the
ferric heme iron in the Met80-oxidized protein, but this coordination
takes place at much lower pH. The ferrous heme iron is ligated by
Met-SO, implying the redox-linked ligand switch in the modified protein.
Binding studies with the model peptide microperoxidase-8 provide a
rationale for alterations in ligation and for the role of the polypeptide
packing in native and Met-SO cyt <i>c</i>. Imidazole binding
experiments have revealed that Lys dissociation from the ferric heme
in K73A/K79G/M80K (M80K<sup>#</sup>) and Met-SO is more than 3 orders
of magnitude slower than the opening of the heme pocket that limits
Met80 replacement in native cyt <i>c</i>. The Lys-to-Met-SO
ligand substitution gates ET of ferric Met-SO cyt <i>c</i> with CoĀ(terpy)<sub>2</sub><sup>2+</sup>. Owing to the slow Lys dissociation
step, ET reaction is slow but possible, which is not the case for
nonswitchable M80A and M80K<sup>#</sup>. Acidic conditions cause Lys
replacement by a water ligand in Met-SO cyt <i>c</i> (p<i>K</i><sub>a</sub> = 6.3 Ā± 0.1), increasing the intrinsic
peroxidase activity of the protein. This pH-driven ligand switch may
be a mechanism to boost peroxidase function of cyt <i>c</i> specifically in apoptotic cells
Remote Perturbations in Tertiary Contacts Trigger Ligation of Lysine to the Heme Iron in Cytochrome <i>c</i>
Perturbations
in protein structure define the mechanism of allosteric
regulation and biological information transfer. In cytochrome <i>c</i> (cyt <i>c</i>), ligation of Met80 to the heme
iron is critical for the proteinās electron-transfer (ET) function
in oxidative phosphorylation and for suppressing its peroxidase activity
in apoptosis. The hard base Lys is a better match for the hard ferric
iron than the soft base Met is, suggesting the key role of the protein
scaffold in favoring Met ligation. To probe the role of the protein
structure in the maintenance of Met ligation, mutations T49V and Y67R/M80A
were designed to disrupt hydrogen bonding and packing of the heme
coordination loop, respectively. Electronic absorption, nuclear magnetic
resonance, and electron paramagnetic resonance spectra reveal that
ferric forms of both variants are Lys-ligated at neutral pH. A minor
change in the tertiary contacts in T49V, away from the heme coordination
loop, appears to be sufficient to execute a change in ligation, suggesting
a cross-talk between the different regions of the protein structure
and a possibility of built-in conformational switches in cyt <i>c</i>. Analyses of thermodynamic stability, kinetics of Lys
binding and dissociation, and the pH-dependent changes in ligation
provide a detailed characterization of the Lys coordination in these
variants and relate these properties to the extent of structural perturbations.
The findings emphasize the importance of the hydrogen-bonding network
in controlling ligation of the native Met80 to the heme iron
Influence of the Interdomain Interface on Structural and Redox Properties of Multiheme Proteins
Multiheme
proteins are important in energy conversion and biogeochemical
cycles of nitrogen and sulfur. A diheme cytochrome c4 (c4) was used as a model
to elucidate roles of the interdomain interface on properties of iron
centers in its hemes A and B. Isolated monoheme domains c4-A and c4-B, together with
the full-length diheme c4 and its Met-to-His
ligand variants, were characterized by a variety of spectroscopic
and stability measurements. In both isolated domains, the heme iron
is Met/His-ligated at pH 5.0, as in the full-length c4, but becomes His/His-ligated in c4-B at higher pH. Intradomain contacts in c4-A are minimally affected by the separation of c4-A and c4-B domains,
and isolated c4-A is folded. In contrast,
the isolated c4-B is partially unfolded,
and the interface with c4-A guides folding
of this domain. The c4-A and c4-B domains have the propensity to interact even without
the polypeptide linker. Thermodynamic cycles have revealed properties
of monomeric folded isolated domains, suggesting that ferrous (FeII), but not ferric (FeIII) c4-A and c4-B, is stabilized by
the interface. This study illustrates the effects of the interface
on tuning structural and redox properties of multiheme proteins and
enriches our understanding of redox-dependent complexation
Becoming a Peroxidase: Cardiolipin-Induced Unfolding of Cytochrome <i>c</i>
Interactions
of cytochrome <i>c</i> (cyt <i>c</i>) with a unique
mitochondrial glycerophospholipid cardiolipin (CL)
are relevant for the proteinās function in oxidative phosphorylation
and apoptosis. Binding to CL-containing membranes promotes cyt <i>c</i> unfolding and dramatically enhances the proteinās
peroxidase activity, which is critical in early stages of apoptosis.
We have employed a collection of seven dansyl variants of horse heart
cyt <i>c</i> to probe the sequence of steps in this functional
transformation. Kinetic measurements have unraveled four distinct
processes during CL-induced cyt <i>c</i> unfolding: rapid
protein binding to CL liposomes; rearrangements of protein substructures
with small unfolding energies; partial insertion of the protein into
the lipid bilayer; and extensive protein restructuring leading to
āopenā extended structures. While early rearrangements
depend on a hierarchy of foldons in the native structure, the later
process of large-scale unfolding is influenced by protein interactions
with the membrane surface. The opening of the cyt <i>c</i> structure exposes the heme group, which enhances the proteinās
peroxidase activity and also frees the C-terminal helix to aid in
the translocation of the protein through CL membranes
Origin of the Conformational Heterogeneity of Cardiolipin-Bound Cytochrome <i>c</i>
Interactions of cytochrome <i>c</i> (cyt <i>c</i>) with cardiolipin (CL) partially unfold the protein, activating
its peroxidase function, a critical event in the execution of apoptosis.
However, structural features of the altered protein species in the
heterogeneous ensemble are difficult to probe with ensemble averaging.
Analyses of the dye-to-heme distance distributions <i>P</i>(<i>r</i>) from time-resolved FRET (TR-FRET) have uncovered
two distinct types of CL-bound cyt <i>c</i> conformations,
extended and compact. We have combined TR-FRET, fluorescence correlation
spectroscopy (FCS), and biolayer interferometry to develop a systematic
understanding of the functional partitioning between
the two conformations. The two subpopulations are in equilibrium with
each other, with a submillisecond rate of conformational exchange
reflecting the protein folding into a compact non-native state, as
well as protein interactions with the lipid surface. Electrostatic
interactions with the negatively charged lipid surface that correlate
with physiologically relevant changes in CL concentrations strongly
affect the kinetics of cyt <i>c</i> binding and conformational
exchange. A predominantly peripheral binding mechanism, rather than
deep protein insertion into the membrane, provides a rationale for
the general denaturing effect of the CL surface and the large-scale
protein unfolding. These findings closely relate to cyt <i>c</i> folding dynamics and suggest a general strategy for extending the
time window in monitoring the kinetics of folding
Superior Fluorescent Probe for Detection of Cardiolipin
Cardiolipin (CL) is a unique phospholipid
found in mitochondrial
inner membrane. It is a key component for mitochondrial function in
both respiration and apoptosis. The level of CL is an important parameter
for investigating these intracellular events and is a critical indicator
of a number of diseases associated with mitochondrial respiratory
functions. 10-Nonyl acridine orange (NAO) is the only fluorescent
dye currently available for CL detection. However, the performance
of NAO is far from satisfactory in terms of selectivity and sensitivity.
In this work, we report an aggregation-induced emission-active fluorogen,
TTAPE-Me, for CL detection and quantification. With improved sensitivity
and excellent selectivity to CL over other major mitochondrial membrane
lipids, TTAPE-Me could serve as a valuable fluorescent sensor for
CL quantification. The use of TTAPE-Me for the quantification of isolated
mitochondria is also demonstrated