Interaction of ferricytochrome c with liposomes

The binding of ferricytochrome c to liposomes consisting of phosphatidylcholine mixtures with cardiolipin (3:1) or phosphatidylserine (3:1) has been investigated. Experimental data have been analyzed in terms of two-dimensional models of large ligand adsorption. The equilibrium parameters of ferricytochrome c interaction with a phospholipid bilayer are determined.     

Fourier-transform infra-red studies of the alkaline isomerization of mitochondrial cytochrome c and the ionization of carboxylic acids.

The alkaline transitions of tuna and horse ferricytochromes c and the trifluoroacetyl-lysine derivative of horse ferricytochrome c have been studied by Fourier-transform (FT) i.r. spectroscopy. The spectral perturbations resulting from the transition have been interpreted by reference to FT i.r. data on simple carboxylic-acid-containing compounds and a bacterial cytochrome c551 in which a haem propionate ionizes without causing a significant conformational change. The analysis strongly suggests that ionization of a haem propionate of mitochondrial cytochrome c triggers the alkaline conformation change.     

Correlation of acid-induced conformational transition of ferricytochrome <Emphasis Type="Italic">c</Emphasis> with cyanide binding kinetics

A relation between pH-induced conformational transitions of horse heart ferricytochrome c and the kinetics of external ligand coordination to heme iron was investigated by optical spectroscopy, circular dichroism and viscometry. The dependencies of both the association, k (a), and dissociation rate constants of cyanide binding on pH were determined from kinetic measurements. The association rate constant exhibits a bell-shaped form of dependence on pH in the region where this protein unfolds. The maximum of the dependence of k (a) on pH is found to be coincident with the pK values of conformational transitions of ferricytochrome c in solutions with both low and high ionic strengths. This observation is explained in terms of ferricytochrome c unfolding, which is characterized by two processes: the gradual opening of the heme crevice accompanied by the detachment of the axial Met80 and its replacement with a water molecule. The former process enhances the rate, whereas the latter results in the inhibition of the rate of cyanide binding.     

Stabilization of the globular structure of ferricytochrome c by chloride in acidic solvents.

Increasing concentrations of chloride were found to increase the resolution between two visible absorbance spectral transitions associated with acidification of ferricytochrome c. Analysis of a variety of spectral and viscosity measurements indicates that protonation of a single group having an apparent pK of 2.1 +/- 0.2 and an intrinsic pK of about 5.3 displaces the methionine ligand without significantly perturbing the native globular conformation. Analysis of methylated ferricytochrome c suggests that protonation of a carboxylate ion, most likely a heme propionate residue, is responsible for displacement of the methionine ligand. Addition of a proton to a second group having an apparent pK of 1.2 +/- 0.1 displaces the histidine ligand and unfolds the protein from a globular conformation into a random coil. It is most likely that the second protonation occurs on the imidazole ring of the histidine ligand itself. Chloride is proposed to perturb these transitions by ligation in the fifth coordination position of the heme ion. Such ligation stabilizes a globular conformation of ferricytochrome c at pH 0.0 and 25 degrees.     

A spin label study of conformational changes in cytochrome c

Spin-labeled pig heart cytochromes c singly modified at Met-65, Tyr-74 and at one of the lysine residues, Lys-72 or Lys-73, were investigated by the ESR method under conditions of different ligand and redox states of the heme and at various pH values. Replacement of Met-80 by the external ligand, cyanide, was shown to produce a sharp increase in the mobility of all the three bound labels while reduction of the spin-labeled ferricytochromes c did not cause any marked changes in their ESR spectra. In the pH range 6-13, two conformational transitions in ferricytochrome c were observed which preceded its alkaline denaturation: the first with pK 9.3 registered by the spin label at the Met-65 position, and the second with pK 11.1 registered by the labels bound to Tyr-74 and Lys-72(73). The conformational changes in the 'left-hand part' of ferricytochrome c are most probably induced in both cases by the exchange of internal protein ligands at the sixth coordination site of the heme.     

The effect of complex formation upon the reduction rates of cytochrome c and cytochrome c peroxidase compound II

The effect of complex formation between ferricytochrome c and cytochrome c peroxidase (Ferrocytochrome-c:hydrogen peroxide oxidoreductase, EC 1.11.1.5) on the reduction of cytochrome c by N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD), reduced N-methylphenazonium methosulfate (PMSH), and ascorbate has been determined at low ionic strength (pH 7) and 25 degrees C. Complex formation with the peroxidase enhances the rate of ferricytochrome c reduction by the neutral reductants TMPD and PMSH. Under all experimental conditions investigated, complex formation with cytochrome c peroxidase inhibits the ascorbate reduction of ferricytochrome c. This inhibition is due to the unfavorable electrostatic interactions between the ascorbate dianion and the negatively charged cytochrome c-cytochrome c peroxidase complex. Corrections for the electrostatic term by extrapolating the data to infinite ionic strength suggest that ascorbate can reduce cytochrome c peroxidase-bound cytochrome c faster than free cytochrome c. Reduction of cytochrome c peroxidase Compound II by dicyanobis(1,10-phenanthroline)iron(II) (Fe(phen)2(CN)2) is essentially unaffected by complex formation between the enzyme and ferricytochrome c at low ionic strength (pH 6) and 25 degrees C. However, reduction of Compound II by the negatively changed tetracyano-(1,10-phenanthroline)iron(II) (Fe(phen)(CN)4) is enhanced in the presence of ferricytochrome c. This enhancement is due to the more favorable electrostatic interactions between the reductant and cytochrome c-cytochrome c peroxidase Compound II complex then for Compound II itself. These studies indicate that complex formation between cytochrome c and cytochrome c peroxidase does not sterically block the electron-transfer pathways from these small nonphysiological reductants to the hemes in these two proteins.     

Raman spectroscopic studies of dimyristoylphosphatidic acid and its interactions with ferricytochrome c in cationic binary and ternary lipid-protein complexes.

The vibrational Raman spectra of both pure 1-alpha-dimyristoylphosphatidic acid (DMPA) liposomes and DMPA multilayers reconstituted with ferricytochrome c at pH 7 and pH 4, with either sodium or calcium as the cation, are reported as a function of temperature. Multilayers composed of a 1:1 mol ratio DMPA and dimyristoylphosphatidylcholine with perdeuterated acyl chains (DMPC-d54) have also been reconstituted with approximately 10(-4) M ferricytochrome c for Raman spectroscopic observation. Total integrated band intensities and relative peak height intensity ratios, two spectral Raman scattering parameters used to characterize bilayer properties, are sensitive to the presence of both ferricytochrome c and the cation in the reconstituted liposomes. Temperature profiles, derived from the various Raman intensity parameters for the 3,100-2,800 cm-1 lipid acyl chain C-H stretching mode region specifically reflect bilayer perturbations due to the interactions of ferricytochrome c. At pH 4 the calcium DMPA multilamellar gel to liquid crystalline phase transition temperatures Tm, defined by either the C-H stretching mode I2850/I2880 and I2935/I2880 peak height intensity ratios, are 58.5 +/- 0.5 degrees C and 60.0 +/- 0.3 degrees C, respectively. This difference in Tm's resolves the phase transition process into first an expansion of the lipid lattice and then a melting of the lipid acyl chains. At pH 7 the calcium DMPA liposomes show no distinct phase transition characteristics below 75 degrees C. For sodium DMPA liposomes reconstituted with ferricytochrome c at either pH 4.0 or pH 7.0, spontaneous Raman spectra show altered lipid structures at temperatures above 40 degrees C. Resonance Raman spectra indicate that ferricytochrome c reconstituted in either calcium or sodium DMPA liposomes changes irreversibly above Tm. For either the binary lipid or ternary lipid-protein systems reconstituted with DMPC-d54, linewidth parameters of the DMPC-d54 acyl chain CD2 symmetric stretching modes at 2,103 cm-1 provide a sensitive measure of the conformational and dynamic properties of the perdeuterated lipid component, while the 3,000 cm-1 C-H spectral region reflects the bilayer characteristics of the DMPA species in the complex. Although calcium clearly induces a lateral phase separation in the DMPA/DMPC-d54 system at pH 7.5 (Kouaouci, R., J.R. Silvius, I. Grah, and M. Pezolet. 1985. Biochemistry. 24:7132-7140), no distinct lateral segregation of the lipid components is observed in the mixed DMPA/DMPC-d54 lipid system in the presence of either ferricytochrome c or the sodium and calcium cations at pH 4.0.(ABSTRACT TRUNCATED AT 400 WORDS)     

Proton NMR comparison of noncovalent and covalently cross-linked complexes of cytochrome c peroxidase with horse, tuna, and yeast ferricytochromes c.

Proton NMR spectroscopy at 500 and 361 MHz has been used to characterize the noncovalent or electrostatic complexes of yeast cytochrome c peroxidase (CcP) with horse, tuna, yeast isozyme-1, and yeast isozyme-2 ferricytochromes c and the covalently cross-linked complexes of cytochrome c peroxidase with horse and yeast isozyme-1 ferricytochromes c. Under the conditions employed in this work, the stoichiometry of the predominant complex formed in solution (which totaled greater than 90% of complex formed) was found to be 1:1 in all cases. These studies have elucidated significant differences in the proton NMR absorption spectra and the one-dimensional nuclear Overhauser effect difference spectra of the complexes, depending on the specific species of ferricytochrome c incorporated. In particular, the results indicate that the noncovalent complexes formed between CcP and physiological redox partners (yeast isozyme-1 or yeast isozyme-2 ferricytochromes c) are distinctly different from the noncovalent complexes formed between CcP and ferricytochromes c from horse and tuna. Parallel chemical cross-linking studies carried out using mixtures of cytochrome c peroxidase with horse ferricytochrome c, and cytochrome c peroxidase with yeast isozyme-1 ferricytochrome c further emphasize such cytochrome c-dependent differences, with only the covalently cross-linked complex of physiological redox partners (cytochrome c peroxidase/yeast isozyme-1) displaying NMR spectra characteristic of a heterogeneous mixture of different 1:1 complexes. Finally, one-dimensional nuclear Overhauser effect experiments have proven valuable in selectively and efficiently probing the protein-protein interface in these complexes, including the environment around the cytochrome c heme 3-methyl group and Phe-82.     

Hydrogen-isotope exchange of oxidized and reduced cytochrome c. A comparison of mass spectrometry and infrared methods.

Hydrogen-deuterium exchange in 2H20 solutions of the two redox states of horse heart cytochrome c was investigated at 20 degrees C, pH 7, by mass spectrometry and infrared spectroscopy. Mass spectrometry indicates that ferricytochrome has 20 hydrogens unexchanged after 24 h, 28 hydrogens exchanging between 10 min and 24 h, and 156 hydrogens exchanging within 10 min; comparative values for ferrocytochrome are 45, 19 and 140. The displacement of the exchange curves obtained by infrared corresponds to 8 to 9 peptide hydrogens. These combined methods show many non-peptide hydrogens exchanging rapidly (87 and 79 for ferricytochrome c and ferrocytochrome c respectively), whereas others, probably buried inside the molecule and involved in hydrogen bonds, are not exchanged, even after 24 h (14 and 30 hydrogens respectively, which is relatively large for a small protein). Infrared results are given in terms of changes of standard free energy for the transconformational reaction which exposes the peptide hydrogens to solvent: in ferricytochrome c and ferrycoytochrome c, 30% and 40% respectively of the peptide hydrogens are protected by conformational transitions stabilized by more than 5 kcal/mol (21 kJ/mol), which implies a large increase in rigidity for the reduced form.     

Nuclear-magnetic-resonance studies of eukaryotic cytochrome c. Assignment of resonances of aromatic amino acids

The aromatic regions of the nuclear magnetic resonance spectra of horse ferricytochrome c and horse ferrocytochrome c are described. Resonance assignments have been made using NMR double-resonance techniques, spectral comparison of related proteins, the perturbing effects of extrinsic probes, and from knowledge of the X-ray structure of cytochrome c. 33 resonances arising from 39 aroumatic protons of ferrocytochrome c, and 18 resonances arising from 27 aromatic protons of ferricytochrome c have been assigned.     

Solution structure of mitochondrial cytochrome c. II. 1H nuclear magnetic resonance of ferrocytochrome c

The 1H nuclear magnetic resonance spectrum of tuna ferrocytochrome c has been studied and the resonances of all 49 amino acid methyl groups have been assigned to specific absorption lines. In comparison with resonance assignments in the ferricytochrome c spectrum, the secondary shifts of resonances of ferrocytochrome c are smaller and the identification of characteristic spin-systems from comparison of spectra from homologous proteins more difficult. For this reason, two-dimensional nuclear magnetic resonance exchange correlated spectroscopy has been used to correlate the assigned resonances of tuna ferricytochrome c with previously unassigned resonances of tuna ferrocytochrome c.     

Conformation of horse heart ferricytochrome c. III. Comparative optical rotatory dispersion study of the protein with its derivative heme undecapeptide

The optical rotatory dispersion of horse heart ferricytochrome c and of a ferri heme undecapeptide have been determined under various conditions. Analysis of the Soret region makes it possible to characterize three different states of ferricytochrome c. the native state (superposition of a negative and a positive Cotton effect); an intermediate state (single positive Cotton effect whose magnitude Δ[M] is equal to 55,000); a denatured state (single positive Cotton effect whose magnitude Δ[M] is equal to 115,000) in which compared to both the native and intermediate states a more or less important decrease in helix content is observed. The optical rotatory dispersion spectra of the Soret region of the monomeric ferri heme undecapeptide is similar to that of denatured ferricytochrome c. The multiplicity of Cotton effects observed under certain conditions for the hemopeptide is a consequence, resulting from a polymerization, of intermolecular interactions. The comparison of the optical rotatory dispersion spectra of ferricytochrome c and the ferri heme undecapeptide indicates that in the intermediate state interactions remain between the heme group and the portion of the poly pep tide chain absent in the hemopeptide. These interactions disappear in the denatured state.     

Low-temperature electron paramagnetic resonance study of the ferricytochrome c-cardiolipin complex

The electron paramagnetic resonance spectrum of the ferricytochrome c complex with cardiolipin was observed at temperatures below 20 K. For the low-spin iron(III) heme system complexed with the negatively charged lipid, the tetragonal and rhombic ligand field parameters (delta/lambda = 3.58, V/lambda = 1.82) differ significantly from those (delta/lambda = 2.53, V/lambda = 1.49) of the free ferricytochrome c sample. The g values of the complex (gx = 1.54 +/- 0.02, gy = 2.26 +/- 0.01, gz = 3.02 +/- 0.01) are compared to the values for free ferricytochrome c (gx = 1.25 +/- 0.02, gy = 2.25 +/- 0.01, gz = 3.04 +/- 0.01). Spectral alterations are interpreted in terms of the ligand field changes induced within the heme group by association with the negatively charged phosphoglyceride.     

Studies on electron transfer from methanol dehydrogenase to cytochrome cL, both purified from Hyphomicrobium X.

Ferricytochrome cL isolated from Hyphomicrobium X is an electron acceptor in assays for homologous methanol dehydrogenase (MDH), albeit a poor one compared with artificial dyes. The intermediates of MDH seen during the reaction are identical with those observed with Wurster's Blue as electron acceptor, indicating that the reaction cycles are similar. The assay showed a pH optimum of approx. 7.0 and scarcely any stimulation by NH4Cl, this being in contrast with assays with artificial dyes, where strong activation by NH4Cl and much higher pH optima have been reported. From the results obtained with stopped-flow as well as steady-state kinetics, combined with the isotope effects found for C2H3OH, it appeared that the dissimilarities between the electron acceptors can be explained from different rate-limiting steps in the reaction cycles. Ferricytochrome cL is an excellent oxidant of the reduced MDH forms at pH 7.0, but the substrate oxidation step is very slow and the activation by NH4Cl is very poor at this pH. At pH 9.0 the reverse situation exists: ferricytochrome cL is a poor oxidant of the reduced forms of MDH at this pH. No C2H3OH isotope effect was observed under these conditions, indicating that substrate oxidation is not rate-limiting, so that activation by NH4Cl cannot be found. Since just the opposite holds for assays with artificial dyes, the poor electron-acceptor capability and the different pH optimum of ferricytochrome cL as well as the insignificant activating effect of NH4Cl (all compared with artificial assays) can be explained. Although different views have been reported on the rate-limiting steps in the systems from Methylophilus methylotrophus and Methylobacterium sp. strain AM1, these are most probably incorrect, as rate-limiting electron transfer between ferrocytochrome cL and horse heart ferricytochrome c can occur. Therefore the conclusions derived for the Hyphomicrobium X system might also apply to the systems from other methylotrophic bacteria. Comparison of the assays performed in vitro (at pH 7.0) having ferricytochrome cL and Wurster's Blue as electron acceptor with methanol oxidation by whole cells shows that the former has similarity whereas the latter has not, this being although ferricytochrome cL is a poor electron acceptor in the assay performed in vitro. The reason for this is the absence of a (natural) activator able to activate the (rate-limiting) substrate oxidation step at physiological pH values.     

Nuclear-magnetic-resonance studies of eukaryotic cytochrome c. Assignment of resonances of aliphatic amino acids

The aliphatic regions of the nuclear magnetic resonance spectra of horse ferricytochrome c and horse ferrocytochrome c are described. Resonance assignments have been made using NMR double-resonance techniques, spectral comparison of related proteins, the perturbing effects of extrinsic probes, and from knowledge of the X-ray structure of cytochrome c. There are eight firmly assigned methyl resonances of ferrocytochrome c and seven firmly assigned methyl resonances of ferricytochrome c.     

Mechanism of resistance of Pediococcus cerevisiae strains to 5-fluorodeoxyuridine.

The kinetics of the reaction of OH radicals with ferricytochrome c was studied in the time range 1 microsecond to 1 s by means of pulse radiolysis. The OH radicals reduce ferricytochrome c by 40% +/- 10%. The time course of the reduction is explained by a mechanism whereby a radical formed after hydrogen has been abstracted from the outer surface of the protein reduces the iron by electron tunnelling. We have calculated that the reducing electron in the radical is bound with an energy of at least 1.75 eV and that the frequency factor of the tunnelling process is v=10(11.5)s-1. This model accounts for the observed absorbance change in time range 5 . 10(-6)--10(-1)s. The time course of the reduction of ferricytochrome c by H radicals (Lichtin, N.N., Shafferman A. and Stein, G. (1974) Biochim. Biophys. Acta 357, 386--398) is explained by the same model.     

Kinetic studies on isomerization of ferricytochrome c in alkaline and acid pH ranges by the circular dichroism stopped-flow method

The isomerization of horse-heart ferricytochrome c caused by varying pH was kinetically studied by using circular dichroism (CD) and optical absorption stopped-flow techniques. In the pH range of 7--13, the existence of the three different forms of ferricytochrome c (pH less than 10, pH 10--12, and pH greater than 12) was indicated from the statistical difference CD spectra. On the basis of analyses of the stopped-flow traces in the near-ultraviolet and Soret wavelength regions, the isomerization of ferricytochrome c from neutral form to the above three alkaline forms was interpreted as follows (1) below pH 10, the replacement of the intrinsic ligand of methionine residue by lysine residue occurs; (2) between pH 10 and 12, the uncoupling of the polypeptide chain from close proximity of the heme group occurs first, followed by the interconversion of the intrinsic ligands; and (3) above pH 12, hydroxide form of ferricytochrome c is formed, though the replacement of the intrinsic ligand by extrinsic ligands may occur via different routes from those below pH 12. The CD changes at 288 nm and in the Soret region caused by the pH-jump (down) from pH 6.0 to 1.6 were compared with the appearance of the 620-nm absorption band ascribed to the formation of the high-spin form of ferricytochrome c. Both CD and absorption changes indicated that the isomerization at pH 1.6 consisted of two processes: one proceeded within the dead-time (about 2 ms) of the stopped-flow apparatus and the other proceeded at a determinable rate with the apparatus. On the basis of these results, the isomerization of ferricytochrome c at pH 1.6 was explained as follows: (1) the transition from the low-spin form to the high-spin forms occurs within about 2 ms, the dead-time of the stopped-flow apparatus; and (2) the polypeptide chain is unfolded after the formation of the high-spin form.     

Polyanion binding to cytochrome c probed by resonance Raman spectroscopy

The interaction of ferricytochrome c with negatively charged heteropolytungstates was studied by resonance Raman spectroscopy. In analogy to previous findings on ferricytochrome c bound to other types of charged interface (Hildebrandt, P. and Stockburger, M. (1989) Biochemistry 28, 6710-6721, 6722-6728), it was shown that in these complexes the conformational states I and II are stabilized. While in state I, the structure is the same as is in the uncomplexed heme protein, in state II three different coordination configurations coexist, i.e., a six-coordinated low-spin, a five-coordinated high-spin and a six-coordinated high-spin form. These configurations constitute thermal coordination equilibria whose thermodynamic properties were determined. The detailed analysis of the low-frequency resonance Raman spectra reveals that in state II the heme pocket assumes an open structure leading to a significantly higher flexibility of the heme group compared to the native ferricytochrome c. It is concluded that these structural changes are the result of Coulombic attractions between the polyanions and the lysine residues around the exposed heme edge which destabilize the heme crevice. Modifications of these interactions upon variation of the ionic strength, the pH or the type of the polytungstate are sensitively reflected by changes of the coordination equilibria in state II as well as of the conformational equilibrium of state I and state II. The conformational changes in state II significantly differ from those associated with the alkaline transition of ferricytochrome c. However, there are some structural similarities between the acid form of the heme protein stable below pH 2.5 in aqueous solution and the six-coordinated high-spin configuration of the bound ferricytochrome c at neutral pH (state II). This suggests that electrostatic interactions with the heteropolytungstates perturb the ionic equilibria of those amino acid side chains which are involved in the acid-induced transition leading to a significant upshift of the apparent pKa.