Reversible unfolding of cytochrome c upon interaction with cardiolipin bilayers. 2. Evidence from phosphorus-31 NMR measurements.

31P NMR measurements were conducted to determine the structural and chemical environment of beef heart cardiolipin when bound to cytochrome c. 31P NMR line shapes infer that the majority of lipid remains in the bilayer state and that the average conformation of the lipid phosphate is not greatly affected by binding to the protein. An analysis of the spin-lattice (T1) relaxation times of hydrated cardiolipin as a function of temperature describes a T1 minimum at around 25 degrees C which leads to a correlation time for the phosphates in the lipid headgroup of 0.71 ns. The relaxation behavior of the protein-lipid complex was markedly different, showing a pronounced enhancement in the phosphorus spin-lattice relaxation rate. This effect of the protein increased progressively with increasing temperature, giving no indication of a minimum in T1 up to 75 degrees C. The enhancement in lipid phosphorus T1 relaxation was observed with protein in both oxidation states, being somewhat less marked for the reduced form. The characteristics of the T1 effects and the influence of the protein on other relaxation processes determined for the lipid phosphorus (spin-spin relaxation and longitudinal relaxation in the rotating frame) point to a strong paramagnetic interaction from the protein. A comparison with the relaxation behavior of samples spinning at the "magic angle" was also consistent with this mechanism. The results suggest that cytochrome c reversibly denatures on complexation with cardiolipin bilayers, such that the electronic ground state prevailing in the native structure of both oxidized and reduced protein can convert to high-spin states with greater magnetic susceptibility.     

Cytochrome c interactions with cardiolipin in bilayers: a multinuclear magic-angle spinning NMR study.

The influence of cytochrome c binding to cardiolipin bilayers on the motional characteristics of each component has been analyzed by magic-angle spinning (MAS) NMR. Observations were made by NMR of natural abundance 31P, 13C, and 1H nuclei in the lipid as well as sites enriched with 13C in the protein. Analysis of methyl carbons enriched in ([epsilon-13CH3]methionine)cytochrome c at residues 65 and 80 reveal quite different behavior for these sites when the protein was bound at a 1:15 molar ratio with hydrated cardiolipin. Cross-polarization (CP) shows a single broad resonance downfield in the methyl region which corresponds to the spectral characteristics of methionine 65 in the solution protein when subjected to moderate thermal perturbations. These observations suggest that although methionine 65 remains motionally restricted when the protein binds to the lipid bilayers, this residue becomes less shielded and exposed to more chemically distinct environments than in the native state of the protein. In contrast to its behavior in native oxidized protein, the methionine 80 methyl could be detected following direct pi/2 pulse excitation, and this residue is assumed to be released from the axial ligand site on the heme iron to become more exposed and highly mobile in the protein-lipid complex. An analysis of the CP response for natural abundance 13C nuclei in the lipid reveals a general increase in motions with slower rates (tens of kilohertz) on binding with cytochrome c, except for sites within the region of fatty acyl chain unsaturation which appear to be selectively mobilized in the complex with protein. It is concluded that, aside from effects on the unsaturated segments, the bound protein induces new modes of slow motions in the lipid assemblies rather than restricting the overall reorientation freedom of the lipid. The strong paramagnetic effects observed previously on the relaxation of phosphorus in protein-bound lipid [Spooner, P.J.R., & Watts, A. (1991) Biochemistry 30, 3880-3885] were not extended to any carbon and proton sites observable by MAS NMR in the lipid, and this infers a specific interaction of lipid phosphate groups with the heme. However, when protein was bound to cardiolipin mixed at a 1:4 mole ratio with dioleoylphosphatidylcholine in bilayers, no direct interaction with the heme was apparent from the phosphorus NMR relaxation behavior in this component, resolved by MAS. Instead, the spectral anisotropy of cardiolipin phosphorus was determined to be reduced, indicating that, on binding with cytochrome c, the headgroup organization was perturbed in this component.(ABSTRACT TRUNCATED AT 400 WORDS)     

Electrostatic interaction of cytochrome c with cytochrome c1 and cytochrome oxidase

The reactions of horse heart cytochrome c with succinate-cytochrome c reductase and cytochrome oxidase were studied as a function of ionic strength using both spectrophotometric and oxygen electrode assay techniques. The kinetic parameter Vmax/Km for both reactions decreased very rapidly as the ionic strength was increased, indicating that electrostatic interactions were important to the reactions. A new semiempirical relationship for the electrostatic energy of interaction between cytochrome c and its oxidation-reduction partners was developed, in which specific complementary charge-pair interactions between lysine amino groups on cytochrome c and negatively charged carboxylate groups on the other protein are assumed to dominate the interaction. The contribution of individual cytochrome c lysine amino groups to the electrostatic interaction was estimated from the decrease in reaction rate caused by specific modification of the lysine amino groups by reagents that change the charge to 0 or -1. These estimates range from -0.9 kcal/mol for lysines immediately surrounding the heme crevice of cytochrome c to 0 kcal/mol for lysines well removed from the heme crevice region. The semiempirical relationship for the total electrostatic energy of interaction was in quantitative agreement with the experimental ionic strength dependence of the reaction rates when the parameters were based on the specific lysine modification results. The electrostatic energies of interaction between cytochrome c and its reductase and oxidase were nearly the same, providing additional evidence that the two reactions take place at similar sites on cytochrome c.     

Loss of molecular interaction between cytochrome c and cardiolipin due to lipid peroxidation.

To explore the molecular mechanism underlying the translocation of cytochrome c from the mitochondrial inner membrane to the cytosol during apoptosis, we analyzed the molecular interaction between cytochrome c and cardiolipin (CL) by (1)H NMR spectroscopy. Bovine heart CL induced a drastic broadening of the linewidth of the downfield signals at 31.4 and 34.2 ppm assigned to the heme methyl group-3 and -8, respectively, of horse heart cytochrome c. In contrast, CL mono- and dihydroperoxides were less active in broadening the signals than CL, and CL trihydroperoxides induced almost no broadening of their linewidth. This finding suggests that the peroxidation of CL induces a release of cytochrome c from mitochondria into the cytosol, which release induces apoptosis in the cells.     

Reversible thermal unfolding of thermostable cytochrome c-552.

Reversible thermal denaturation of cytochrome c-552 from the extremely thermophilic bacterium Thermus thermophilus was studied by circular dichroism and fluorescence spectroscopy. Thermal denaturation in the presence of guanidine hydrochloride is completely reversible. The thermodynamic parameters for the reaction have been calculated based on a two-state mechanism. The free energy change on denaturation (ΔG) at 25 °C in the absence of denaturant is estimated to be 28.5 ± 0.15 kcal/mol, which is larger than that of cytochrome c from mesophilic organisms. The temperature of maximum stability is approximately 27 °C, which is higher than those of cytochromes c from mesophilic organisms (9 to 12 °C). The temperature dependences of the enthalpy and entropy changes are similar to those of cytochromes c from mesophilic organisms. The heat capacity change on denaturation is between 1250 and 1680 cal/deg mole, which is similar to those of cytochromes c from mesophilic organisms (1500 to 2500 cal/deg mol). From these results, it has been concluded that T. thermophilus cytochrome c is more stable than cytochromes c from mesophilic organisms by virtue of the fact that the free energy change for denaturation is greater and has its maximum at a higher temperature.     

Solution NMR study of the yeast cytochrome c peroxidase: cytochrome c interaction

Here we present a solution NMR study of the complex between yeast cytochrome c (Cc) and cytochrome c peroxidase (CcP), a paradigm for understanding the biological electron transfer. Performed for the first time, the CcP-observed heteronuclear NMR experiments were used to probe the Cc binding in solution. Combining the Cc- and CcP-detected experiments, the binding interface on both proteins was mapped out, confirming that the X-ray structure of the complex is maintained in solution. Using NMR titrations and chemical shift perturbation analysis, we show that the interaction is independent of the CcP spin-state and is only weakly affected by the Cc redox state. Based on these findings, we argue that the complex of the ferrous Cc and the cyanide-bound CcP is a good mimic of the catalytically-active Cc–CcP compound I species. Finally, no chemical shift perturbations due to the Cc binding at the low-affinity CcP site were observed at low ionic strength. We discuss possible reasons for the absence of the effects and outline future research directions.     

Ligand exchange during unfolding of cytochrome c.

The productive folding pathway of cytochrome c passes through an obligatory HW intermediate in which the heme is coordinated by a solvent water molecule and a native ligand, His-18, prior to the formation of the folded HM state with both the native His-18 and Met-80 heme coordination. Two off pathway intermediates, a five-coordinated state (5C) and a bis-histidine state (HH), were also identified during the folding reaction. In the present work, the thermodynamics and the kinetics of the unfolding reaction of cytochrome c were investigated with resonance Raman scattering, tryptophan fluorescence spectroscopy, and circular dichroism. The objective of these experiments was to determine if the protein opens up and diverges into the differing heme ligation states through a many pathway mechanism or if it passes through intermediate states analogous to those observed during the folding reaction. Equilibrium unfolding results indicate that, in contrast to 5C, the stability of HH with respect to HW decreases as the concentration of GdnHCl increases. The difference in their response to the denaturant indicates that the polypeptide structure of 5C is relatively loose as compared with HH in which the polypeptide is misfolded. Time-resolved resonance Raman measurements show that strikingly similar ligand exchange reactions occur during unfolding as were observed during folding. Combined with fluorescence data, a kinetic model is proposed in which local structural rearrangements controlled by heme ligand exchange reactions appear prior to the global relaxation of the polypeptide chain.     

Cytochrome c interaction with membranes. Formylated cytochrome c.

A single species of tryptophan-59 formylated cytochrome c with a half-reduction potential of 0.085 ± 0.01 V at pH 7.0 was used to study its catalytic and functional properties. The spectral properties of the modified cytochrome show that the 6th ligand position is open to reaction with azide, cyanide, and carbon monoxide. Formylated cytochrome c binds to cytochrome c depleted rat liver and pigeon heart mitochondria with the precise stoichiometry of two modified cytochrome c molecules per molecule of cytochrome a (K_D of approx 0.1 μm). Formylated cytochrome c was reducible by ascorbate and was readily oxidized by cytochrome c oxidase. The apparent K_m value of the oxidase for the formylated cytochrome c was six times higher than for the native cytochrome and the apparent V was smaller. Formylated cytochrome c does not restore the oxygen uptake in C-depleted mitochondria but inhibits, in a competitive manner, the oxygen uptake induced by the addition of native cytochrome c. Formylated cytochrome c was inactive in the reaction with mitochondrial NADH-cytochrome c reductase but was able to accept electrons through the microsomal NADPH-cytochrome c reductase.     

Equilibrium unfolding of a small low-potential cytochrome, cytochrome c553 from Desulfovibrio vulgaris.

To understand general aspects of stability and folding of c-type cytochromes, we have studied the folding characteristics of cytochrome c553 from Desulfovibrio vulgaris (Hildenborough). This cytochrome is structurally similar but lacks sequence homology to other heme proteins; moreover, it has an abnormally low reduction potential. Unfolding of oxidized and reduced cytochrome c553 by guanidine hydrochloride (GuHCl) was monitored by circular dichroism (CD) and Soret absorption; the same unfolding curves were obtained with both methods supporting that cytochrome c553 unfolds by an apparent two-state process. Reduced cytochrome c553 is 7(3) kJ/mol more stable than the oxidized form; accordingly, the reduction potential of unfolded cytochrome c553 is 100(20) mV more negative than that of the folded protein. In contrast to many other unfolded cytochrome c proteins, upon unfolding at pH 7.0 both oxidized and reduced heme in cytochrome c553 become high-spin. The lack of heme misligation in unfolded cytochrome c553 implies that its unfolded structure is less constrained than those of cytochromes c with low-spin, misligated hemes.     

Nitric oxide inhibits peroxidase activity of cytochrome c.cardiolipin complex and blocks cardiolipin oxidation

The increased production of NO during the early stages of apoptosis indicates its potential involvement in the regulation of programmed cell death through yet to be identified mechanisms. Recently, an important role for catalytically competent peroxidase form of pentacoordinate cytochrome c (cyt c) in a complex with a mitochondria-specific phospholipid, cardiolipin (CL), has been demonstrated during execution of the apoptotic program. Because the cyt c.CL complex acts as CL oxygenase and selectively oxidizes CL in apoptotic cells in a reaction dependent on the generation of protein-derived (tyrosyl) radicals, we hypothesized that binding and nitrosylation of cyt c regulates CL oxidation. Here we demonstrate by low temperature electron paramagnetic resonance spectroscopy that CL facilitated interactions of ferro- and ferri-states of cyt c with NO and NO(-), respectively, to yield a mixture of penta- and hexa-coordinate nitrosylated cyt c. In the nitrosylated cyt c.CL complex, NO chemically reacted with H(2)O(2)-activated peroxidase intermediates resulting in their reduction. A dose-dependent quenching of H(2)O(2)-induced protein-derived radicals by NO donors was shown using direct electron paramagnetic resonance measurements as well as immuno-spin trapping with antibodies against protein 5,5-dimethyl-1-pyrroline N-oxide-nitrone adducts. In the presence of NO donors, H(2)O(2)-induced oligomeric forms of cyt c positively stained for 3-nitrotyrosine confirming the reactivity of NO toward tyrosyl radicals of cyt c. Interaction of NO with the cyt c.CL complex inhibited its peroxidase activity with three different substrates: CL, etoposide, and 3,3'-diaminobenzidine. Given the importance of CL oxidation in apoptosis, mass spectrometry analysis was utilized to assess the effects of NO on oxidation of 1,1'2,2'-tertalinoleoyl cardiolipin. NO effectively inhibited 1,1'2,2'-tertalinoleoyl cardiolipin oxidation catalyzed by the peroxidase activity of cyt c. Thus, NO can act as a regulator of peroxidase activity of cyt c.CL complexes.     

Two-dimensional 1H NMR studies of cytochrome c

Two-dimensional nuclear magnetic resonance techniques were used to assign the NH, C alpha H, and C beta H protons of over 60 of the 104 amino acid residues in the 1H NMR spectrum of horse ferrocytochrome c. The majority of these amino acids were completely assigned. Assignments were based on the analysis of two-dimensional J-correlated (COSY), nuclear Overhauser effect (NOESY), and relayed COSY spectra and on comparisons of the J-correlated spectra of various cytochrome c species. Spin diffusion is not a problem with monomeric proteins the size of cytochrome c. Here these advances are illustrated with data that lead to the assignment of the heme-associated residues cysteine-14 and tryptophan-59, the axial ligands methionine-80 and histidine-18, the entire N-terminal helix, and several other amino acid spin systems. With these approaches, structure, structure change, the internal dynamics of cytochrome c, and the interaction of these with function are being studied, especially by observation of the hydrogen exchange behavior of essentially all the H-bonded amides and some side chain protons in both the reduced and oxidized proteins.     

Calcium-induced release of cytochrome c from cardiolipin nanodisks: Implications for apoptosis

Miniature bilayer membranes comprised of phospholipid and an apolipoprotein scaffold, termed nanodisks (ND), have been used in binding studies. When ND formulated with cardiolipin (CL), but not phosphatidylcholine, were incubated with cytochrome c, FPLC gel filtration chromatography provided evidence of a stable binding interaction. Incubation of CL ND with CaCl₂ resulted in a concentration-dependent increase in sample turbidity caused by ND particle disruption. Prior incubation of CL ND with cytochrome c increased CL ND sensitivity to CaCl₂-induced effects. Centrifugation of CaCl₂-treated CL ND samples yielded pellet and supernatant fractions. Whereas the ND scaffold protein, apolipophorin III, was recovered in the pellet fraction along with CL, the majority of the cytochrome c pool was in the supernatant fraction. Moreover, when cytochrome c CL ND were incubated with CaCl₂ at concentrations below the threshold to induce ND particle disruption, FPLC analysis showed that cytochrome c was released. Pre-incubation of CL ND with CaCl₂ under conditions that do not disrupt ND particle integrity prevented cytochrome c binding to CL ND. Thus, competition between Ca²⁺ and cytochrome c for a common binding site on CL modulates cytochrome c binding and likely plays a role in its dissociation from CL-rich cristae membranes in response to apoptotic stimuli.     

Reversible independent unfolding of the domains of urokinase monitored by 1H NMR

Human urinary-type plasminogen activator (urokinase) and proteolytic fragments corresponding to the kringle, EGF-kringle, and protease domains have been examined by 1H NMR spectroscopy. The intact protein shows a very well-resolved spectrum for a molecule of this size (MW 54,000), with resonance line widths not greatly increased from those of the isolated domains. On increasing the temperature, the protein at pH values close to 4 was found to undergo two distinct and reversible conformational transitions. These were identified, by comparison with spectra of the proteolytic fragments, as the unfolding of the kringle (and EGF) domains (at approximately 42 degrees C) and of a segment of the protease domain (at approximately 60 degrees C). The remaining segment of the protease domain showed persistent structure to at least 85 degrees C at pH 4; only at lower pH values could complete unfolding be achieved. The results indicate that the structures and stabilities of the isolated domains are closely similar to those in the intact protein and suggest that there is a degree of independent motion at least between the kringle and protease domains.     

Characterization of four covalently-linked yeast cytochrome c/cytochrome c peroxidase complexes: Evidence for electrostatic interaction between bound cytochrome c molecules

Four covalent complexes between recombinant yeast cytochrome c and cytochrome c peroxidase (rCcP) were synthesized via disulfide bond formation using specifically designed protein mutants (Papa, H. S., and Poulos, T. L. (1995) Biochemistry 34, 6573-6580). One of the complexes, designated V5C/K79C, has cysteine residues replacing valine-5 in rCcP and lysine-79 in cytochrome c with disulfide bond formation between these residues linking the two proteins. The V5C/K79C complex has the covalently bound cytochrome c located on the back-side of cytochrome c peroxidase, approximately 180 degrees from the primary cytochrome c-binding site as defined by the crystallographic structure of the 1:1 noncovalent complex (Pelletier, H., and Kraut J. (1992) Science 258, 1748-1755). Three other complexes have the covalently bound cytochrome c located approximately 90 degrees from the primary binding site and are designated K12C/K79C, N78C/K79C, and K264C/K79C, respectively. Steady-state kinetic studies were used to investigate the catalytic properties of the covalent complexes at both 10 and 100 mM ionic strength at pH 7.5. All four covalent complexes have catalytic activities similar to those of rCcP (within a factor of 2). A comprehensive study of the ionic strength dependence of the steady-state kinetic properties of the V5C/K79C complex provides evidence for significant electrostatic repulsion between the two cytochromes bound in the 2:1 complex at low ionic strength and shows that the electrostatic repulsion decreases as the ionic strength of the buffer increases.     

1H NMR and ESR studies of oxidized cytochrome c551 from Pseudomonas aeruginosa.

Near neutral pH, Fe(III) cytochrome c551 exhibits an ESR absorption due primarily to a single species with g values of 3.24, 2.06, and 1.48. These g values are somewhat different from those of horse heart cytochrome c and can be interpreted by the generalizations of Brautigan et al. [(1977) J. Biol. Chem. 252, 574] to be due to Fe binding by the imidazole anion of histidine rather than by neutral imidazole. The NMR spectrum of Fe(III) cytochrome c551 exhibits a number of hyperfine-shifted peaks whose pattern shows similarities to but many differences from that of horse heart cytochrome c. Variation in shifts of some of the peaks in the pH range 5--9 is ascribed to ionization of a somewhat buried propionic acid side chain (pK = 5.8) and to ionization of the N-terminal NH3+ group (pK = 7.7). At alkaline pH greater than 9.4, as shown by a variety of optical and ESR spectral changes, the Met-61 S ligand is replaced by other ligands.     

Mechanism of peroxynitrite interaction with cytochrome c

Kinetics of the reaction of peroxynitrite with ferric cytochrome c in the absence and presence of bicarbonate was studied. It was found that the heme iron in ferric cytochrome c does not react directly with peroxynitrite. The rates of the absorbance changes in the Soret region of cytochrome c spectrum caused by peroxynitrite or peroxynitrite/bicarbonate were the same as the rate of spontaneous isomerization of peroxynitrite or as the rate of the reaction of peroxynitrite with bicarbonate, respectively. This means that intermediate products of peroxynitrite decomposition, (.)OH/(.)NO(2) or, in the presence of bicarbonate, CO(3)(-)(.)/(.)NO(2), are the species responsible for the absorbance changes in the Soret band of cytochrome c. Modifications of the heme center of cytochrome c by radiolytically produced radicals, (.)OH, (.)NO(2) or CO(3)(-)(.), were also studied. The absorbance changes in the Soret band caused by radiolytically produced (.)OH or CO(3)(-)(.) were much more significant that those observed after peroxynitrite treatment, compared under similar concentrations of radicals. (.)NO(2) produced radiolytically did not interact with the heme center of cytochrome c. Cytochrome c exhibited an increased peroxidase-like activity after reaction with peroxynitrite as well as with radiolytically produced (.)OH, (.)NO(2) or CO(3)(-)(.) radicals. This means that modification of protein structure: oxidation of amino acids and/or tyrosine nitration, facilitates reaction of H(2)O(2) with the heme iron of cytochrome c, followed by reaction with the second substrate.