Peroxidase activity and structural transitions of cytochrome c bound to cardiolipin-containing membranes

During apoptosis, cytochrome c (cyt c) is released from intermembrane space of mitochondria into the cytosol where it triggers the caspase-dependent machinery. We discovered that cyt c plays another critical role in early apoptosis as a cardiolipin (CL)-specific oxygenase to produce CL hydroperoxides required for release of pro-apoptotic factors [Kagan, V. E., et al. (2005) Nat. Chem. Biol. 1, 223-232]. We quantitatively characterized the activation of peroxidase activity of cyt c by CL and hydrogen peroxide. At low ionic strength and high CL/cyt c ratios, peroxidase activity of the CL/cyt c complex was increased >50 times. This catalytic activity correlated with partial unfolding of cyt c monitored by Trp(59) fluorescence and absorbance at 695 nm (Fe-S(Met(80)) band). The peroxidase activity increase preceded the loss of protein tertiary structure. Monounsaturated tetraoleoyl-CL (TOCL) induced peroxidase activity and unfolding of cyt c more effectively than saturated tetramyristoyl-CL (TMCL). TOCL/cyt c complex was found more resistant to dissociation by high salt concentration. These findings suggest that electrostatic CL/cyt c interactions are central to the initiation of the peroxidase activity, while hydrophobic interactions are involved when cyt c's tertiary structure is lost. In the presence of CL, cyt c peroxidase activity is activated at lower H(2)O(2) concentrations than for isolated cyt c molecules. This suggests that redistribution of CL in the mitochondrial membranes combined with increased production of H(2)O(2) can switch on the peroxidase activity of cyt c and CL oxidation in mitochondria-a required step in execution of apoptosis.     

Conductivity and structural transitions in bilayer lipid membranes

5 structural transitions were found in bilayer lipid membranes (BLM) from egg lecithin (EL) within the temperature range 14-44 degrees C. In the transition zone BLM conductivity abruptly increases, in some cases current fluctuations of the order 150 pC of the channel type are initiated. The transition temperatures observed in BLM from EL coincide with those in biological membranes. The cause of this phenomenon is discussed, as well as possible use of these BLM in the region of structural transition as a model of cellular receptor to electromagnetic fields.     

A structural model for the adduct between cytochrome c and cytochrome c oxidase

An ensemble of structural models of the adduct between cytochrome c and cytochrome c oxidase from Paracoccus denitrificans has been calculated based on the experimental data from site-directed mutagenesis and NMR experiments that have accumulated over the last years of research on this system. The residues from each protein that are at the protein–protein interface have been identified by the above experimental work, and this information has been converted in a series of restraints explicitly used in calculations. It is found that a single static structural model cannot satisfy all experimental data simultaneously. Therefore, it is proposed that the adduct exists as a dynamic ensemble of different orientations in equilibrium, and may be represented by a combination or average of the various limiting conformations calculated here. The equilibrium involves both conformations that are competent for electron transfer and conformations that are not. Long-range recognition of the partners is driven by non-specific electrostatic interactions, while at shorter distances hydrophobic contacts tune the reciprocal orientation. Electron transfer from cytochrome bc ₁ to cytochrome c oxidase is mediated through cytochrome c experiencing multiple encounters with both of its partners, only part of which are productive. The number of encounters, and thus the electron transfer rate, may be increased by the formation of a cytochrome bc ₁–cytochrome c oxidase supercomplex and/or (in human) by increasing the concentration of the two enzymes in the membrane space. Protein Data Bank Accession numbers The coordinates of the five best structural models for each of the four clusters have been deposited in the Protein Data Bank (PDB ID 1ZYY).     

Assignment of hyperfine shifted resonances in high-spin forms of cytochrome c peroxidase by reconstitutions with deuterated hemins

There is good evidence to show that ferric enterochelin is an essential growth factor for a number of Gram-negative pathogenic bacteria exposed to the host iron binding proteins, transferrin and lactoferrin. Tests of nineteen complexes of enterochelin as potential antibacterial agents showed that only those containing either indium (In3+) or scandium (Sc3+) inhibited bacterial growth. In this study, further evidence is presented which demonstrates a competition between the Sc3+ and Fe3+ complexes. The uptake of both complexes is energy dependent and is also repressed in iron-replete cells. The Sc3+ complex accumulates within the cells at 20% of the rate of the Fe3+ complex. The main components of the ferric enterochelin transport system are required for the transport of the Sc3+ complex although some Sc3+ appears to enter the cell by another route. The accumulation, within the cell, of 14C-labelled enterochelin complexes depends on the growth medium. The relationship of the size of the metal ion to the biological activity of the complex is discussed and possible mechanisms of action of the Sc3+ complex are considered.     

Protein matrix and dielectric effect in cytochrome c

The effect of the protein matrix on the standard potential of a buried redox center has been investigated by using a selection of mutants and chemical derivatives in Saccharomyces cerevisiae cytochrome c isoform 1. Assuming only local structural perturbation and no alteration of the iron-ligation chemistry, Delta E(m)(0)' can be regarded as a measure of the difference in polypeptide solvation of the heme charge, which reflects the dielectric properties of the protein. The evaluation of an apparent dielectric constant (U(exp)/U(theo)) yields variable, and sometimes even negative, values if U(exp) = Delta G(0)redox. However, some consistent result are observed if U(exp) = Delta H(0)redox, with a measured epsilon(Delta Delta)(H)(redox) = 19 +/- 6. The variability is thus attributed to an entropic factor (epsilon(Delta Delta)(S)(redox)) that is investigated using a series of substitutions of Asn(52) and/or Tyr(67). In double mutants Y67F/N52I Y67F/N52V, where most of the hydrogen bond network in the heme crevice is eliminated, Delta S(redox) compares to the wild type. This indicates that a fully consistent hydrogen bond network has a similar polarizability as an apolar matrix. We therefore argue that the variability in net dielectric susceptibility arises from conformational polarizability, a factor that is not a function of atomic properties and coordinates and is therefore hard to predict using conventional physical relationships.     

Fast structural dynamics in reduced and oxidized cytochrome c

The sub‐nanosecond structural dynamics of reduced and oxidized cytochrome c were characterized. Dynamic properties of the protein backbone measured by amide ¹⁵N relaxation and side chains measured by the deuterium relaxation of methyl groups change little upon change in the redox state. These results imply that the solvent reorganization energy associated with electron transfer is small, consistent with previous theoretical analyses. The relative rigidity of both redox states also implies that dynamic relief of destructive electron transfer pathway interference is not operational in free cytochrome c.     

A kinetic study of the alkaline transitions in cytochrome c peroxidase

Cytochrome c peroxidase undergoes a complex series of transitions between pH 8 and 14. Seven distinct spectral transitions occur between 4 ms and 24 h after exposure to alkaline pH. The fastest transition occurs within the mixing time of a stopped-flow instrument and converts the native high-spin ferric form of the enzyme to a low-spin form which may be the hydroxy complex of the enzyme. An apparent pKa of 9.7 +/- 0.2 relates the native and initial alkaline form of the enzyme. Three other low-spin enzyme forms are evident from the experimental data prior to denaturation of the enzyme and complete exposure of the heme to the solvent. The final denaturation process occurs with an apparent pKa of 10.3 +/- 0.3.     

Differential detergent solubility investigation of thermally induced transitions in cytochrome c oxidase

The thermal denaturation of membrane-reconstituted cytochrome c oxidase (EC 1.9.3.1) occurs at approximately 63 degrees C as determined by high-sensitivity differential scanning calorimetry. The heat capacity profile associated with this process is characterized by the presence of two well-defined peaks, indicating that all the enzyme subunits do not have the same thermal stability. This thermal denaturation of the enzyme complex is coupled to a change in its solubility properties. This change in solubility allows separation of the native and denatured protein fractions by detergent solubilization followed by centrifugation under conditions in which only the native fraction is solubilized. Using this principle, it has been possible to study the denaturation of membrane-reconstituted cytochrome c oxidase and quantitatively identify the protein subunits undergoing thermal denaturation using computer-assisted gel electrophoresis analysis. This technique allows calculation of single-subunit thermal denaturation profiles within the intact enzyme complex, and as such, it can be used to obtain transition temperatures, molecular populations, and van't Hoff enthalpy changes for individual protein subunits, thus complementing results obtained by high-sensitivity differential scanning calorimetry.     

Structural transitions of carboxymethylated cytochrome c: calorimetric and circular dichroic studies

Circular dichroism and differential scanning calorimetry studies on the unfolding-refolding process of native and carboxymethylated cytochrome c, induced either by temperature or chemical agents, have been performed. The results have shown that the modified protein has a decreased conformational stability with respect to the native state, in agreement with a structure less compact, but still highly folded, which behaves as a thermodynamically stable "intermediate" between native and fully unfolded cytochrome c.     

Acetaminophen inhibits cytochrome c redox cycling induced lipid peroxidation

Recent evidence indicates that site-specific crosstalk between O-GlcNAcylation and phosphorylation and the O-GlcNAcylation of kinases play an important role in regulating cell signaling. However, relatively few kinases have been analyzed for O-GlcNAcylation. Here, we identify additional kinases that are substrates for O-GlcNAcylation using an in vitro OGT assay on a functional kinase array. Forty-two kinases were O-GlcNAcylated in vitro, representing 39% of the kinases on the array. In addition, we confirmed the in vivo O-GlcNAcylation of three identified kinases. Our results suggest that O-GlcNAcylation may directly regulate a substantial number of kinases and illustrates the increasingly complex relationship between O-GlcNAcylation and phosphorylation in cellular signaling.     

Variable proton-pumping stoichiometry in structural variants of cytochrome c oxidase

Cytochrome c oxidase is a multisubunit membrane-bound enzyme, which catalyzes oxidation of four molecules of cytochrome c²⁺ and reduction of molecular oxygen to water. The electrons are taken from one side of the membrane while the protons are taken from the other side. This topographical arrangement results in a charge separation that is equivalent to moving one positive charge across the membrane for each electron transferred to O₂. In this reaction part of the free energy available from O₂ reduction is conserved in the form of an electrochemical proton gradient. In addition, part of the free energy is used to pump on average one proton across the membrane per electron transferred to O₂. Our understanding of the molecular design of the machinery that couples O₂ reduction to proton pumping in oxidases has greatly benefited from studies of so called “uncoupled” structural variants of the oxidases. In these uncoupled oxidases the catalytic O₂-reduction reaction may display the same rates as in the wild-type CytcO, yet the electron/proton transfer to O₂ is not linked to proton pumping. One striking feature of all uncoupled variants studied to date is that the (apparent) pK_a of a Glu residue, located deeply within a proton pathway, is either increased or decreased (from 9.4 in the wild-type oxidase). The altered pK_a presumably reflects changes in the local structural environment of the residue and because the Glu residue is found near the catalytic site as well as near a putative exit pathway for pumped protons these changes are presumably important for controlling the rates and trajectories of the proton transfer. In this paper we summarize data obtained from studies of uncoupled structural oxidase variants and present a hypothesis that in quantitative terms offers a link between structural changes, modulation of the apparent pK_a and uncoupling of proton pumping from O₂ reduction.     

The hierarchy of structural transitions induced in cytochrome c by anionic phospholipids determines its peroxidase activation and selective peroxidation during apoptosis in cells

Activation of peroxidase catalytic function of cytochrome c (cyt c) by anionic lipids is associated with destabilization of its tertiary structure. We studied effects of several anionic phospholipids on the protein structure by monitoring (1) Trp59 fluorescence, (2) Fe-S(Met80) absorbance at 695 nm, and (3) EPR of heme nitrosylation. Peroxidase activity was probed using several substrates and protein-derived radicals. Peroxidase activation of cyt c did not require complete protein unfolding or breakage of the Fe-S(Met80) bond. The activation energy of cyt c peroxidase changed in parallel with stability energies of structural regions of the protein probed spectroscopically. Cardiolipin (CL) and phosphatidic acid (PA) were most effective in inducing cyt c peroxidase activity. Phosphatidylserine (PS) and phosphatidylinositol bisphosphate (PIP2) displayed a significant but much weaker capacity to destabilize the protein and induce peroxidase activity. Phosphatidylinositol trisphosphate (PIP3) appeared to be a stronger inducer of cyt c structural changes than PIP2, indicating a role for the negatively charged extra phosphate group. Comparison of cyt c-deficient HeLa cells and mouse embryonic cells with those expressing a full complement of cyt c demonstrated the involvement of cyt c peroxidase activity in selective catalysis of peroxidation of CL, PS, and PI, which corresponded to the potency of these lipids in inducing cyt c's structural destabilization.     

Chemiluminescence of lipid vesicles supplemented with cytochrome c and hydroperoxide.

The increase in light emission of hydroperoxide-supplemented cytochrome c observed on addition of lipid vesicles was related to the degree of unsaturation of the fatty acids of the phospholipids: dipalmitoyl phosphatidylcholine was without effect, whereas dioleoyl phosphatidylcholine and soya-bean phosphatidylcholine enhanced chemiluminescence 2- and 3-fold respectively. Effects on light-emission were similar to those on O2 uptake. The chemiluminescence of the present system was sensitive to cyanide and to the radical trap 2,5-di-t-butylquinol, indicating a catlytic activity of cytochrome c and the presence of free-radical species respectively. Lipid-vesicle enhanced chemiluminescence showed different kinetic behaviours, apparently depending on unsaturation: three phases are described for soya-bean phosphatidylcholine, whereas only one phase was present in mixtures containing dipalmitoyl and dioleoyl phospholipids. Chemiluminescence of lipid vesicles supplemented with cytochrome c and hydroperoxide showed similar kinetic patterns with H2O2 and primary (ethyl) and tertiary (t-butyl and cumene) hydroperoxides. Participation of singlet molecular oxygen, mainly on the phase III of chemiluminescence, is suggested by the increase of light-emission by 1,4-diazabicyclo[2.2.2]-octane as well as by data from spectral analysis.     

Heme-heme communication during the alkaline-induced structural transition in cytochrome c oxidase

Alkaline-induced conformational changes at pH 12.0 in the oxidized as well as the reduced state of cytochrome c oxidase have been systematically studied with time-resolved optical absorption and resonance Raman spectroscopies. In the reduced state, the heme a(3) first converts from the native five-coordinate configuration to a six-coordinate bis-histidine intermediate as a result of the coordination of one of the Cu(B) ligands, H290 or H291, to the heme iron. The coordination state change in the heme a(3) causes the alteration in the microenvironment of the formyl group of the heme a(3) and the disruption of the H-bond between R38 and the formyl group of the heme a. This structural transition, which occurs within 1min following the initiation of the pH jump, is followed by a slower reaction, in which Schiff base linkages are formed between the formyl groups of the two hemes and their nearby amino acid residues, presumably R38 and R302 for the heme a and a(3), respectively. In the oxidized enzyme, a similar Schiff base modification on heme a and a(3) was observed but it is triggered by the coordination of the H290 or H291 to heme a(3) followed by the breakage of the native proximal H378-iron and H376-iron bonds in heme a and a(3), respectively. In both oxidation states, the synchronous formation of the Schiff base linkages in heme a and a(3) relies on the structural communication between the two hemes via the H-bonding network involving R438 and R439 and the propionate groups of the two hemes as well as the helix X housing the two proximal ligands, H378 and H376, of the hemes. The heme-heme communication mechanism revealed in this work may be important in controlling the coupling of the oxygen and redox chemistry in the heme sites to proton pumping during the enzymatic turnover of CcO.