Characterization of the interaction of cytochrome c and mitochondrial ubiquinol-cytochrome c reductase

Characterization of the steady state kinetics of reduction of horse ferricytochrome c by purified beef ubiquinol-cytochrome c reductase, employing 2,3-dimethoxy-5-methyl-6-decylbenzoquinol as reductant, has shown that: 1) the dependence of the reaction on quinol and on ferricytochrome c concentration is consistent with a ping-pong mechanism; 2) the pH optimum of the reaction is near 8.0; 3) the effect of ionic strength on the apparent Km and the TNmax of the reaction for the native cytochrome c is small, and at higher cytochrome c concentrations substrate inhibition is observed; 4) the effect of ionic strength on the kinetic parameters for the reaction of 4-carboxy-2,6-dinitrophenyllysine 27 horse cytochrome c is much larger than for the native protein; and 5) competitive product inhibition is also observed with a Ki consistent with the binding affinity of ferrocytochrome c for Complex III, as determined by gel filtration. In addition, direct binding measurements demonstrated that ferricytochrome c binds more tightly than the reduced protein to Complex III under low ionic strength conditions and that under these conditions more than one molecule of cytochrome c is bound per molecule of Complex III. Exchange of Complex III into a nonionic detergent decreases this excess nonspecific binding. Measurement of the rates of dissociation of the oxidized and reduced 1:1 complexes of cytochrome c and Complex III by stopped flow was consistent with the disparity of binding affinities, the dissociation rate constant for ferrocytochrome c being about 5-fold higher than that for the ferric protein. A model which accounts for the properties of this system is described, assuming that cytochrome c bound to noncatalytic sites on the respiratory complex decreases the catalytic site binding constant for the substrate.     

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.     

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.     

Kinetics of electron transfer between mitochondrial cytochrome c and iron hexacyanides

The reduction of horse and Candida krusei cytochromes c by ferrocyanide has been studied by 1H NMR spectroscopy and the reaction found to involve a precursor complex of ferrocyanide bound to ferricytochrome c (pH* 7.4, 2H2O, I = 0.12, and 25 degrees C). The electron transfer rate constants for the reduction of the two ferricytochromes by associated ferrocyanide were found to be the same at 780 +/- 80 sec-1 but the association constants for binding of ferrocyanide to ferricytochrome c were significantly different: horse, 90 +/- 20 M-1 and Candida, 285 +/- 30 M-1. The different association constants partly accounts for the previously observed reactivity difference between horse and Candida cytochromes c. Comparison of the NMR data with data obtained by other kinetic methods has allowed the electron transfer rate constant for the oxidation of ferrocytochrome c by associated ferricyanide to be determined. This was found to be 4.6 +/- 1 X 10(4) sec-1.     

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.     

The stability of ferricytochrome c. Temperature dependence of its NMR spectrum

The temperature dependence of the nuclear magnetic resonance spectrum of horse ferricytochrome c is described. The protein maintains an ordered structure over the temperature range 20 degrees C to 77 degrees C. The temperature dependence of the spectrum of ferricytochrome c arises from a number of causes including the paramagnetism of the ferric ion and protein structural changes. Preliminary analysis of the data show that the region of the protein about Ile-57 is flexible. Comparison of the data with the analogous data for horse ferrocytochrome c reveals that there is a small difference in structure between cytochrome c in its two oxidation states in the region about Ile-57.     

Proton-NMR studies of the effects of ionic strength and pH on the hyperfine-shifted resonances and phenylalanine-82 environment of three species of mitochondrial ferricytochrome c.

Ferricytochromes c from three species (horse, tuna, yeast) display sensitivity to variations in solution ionic strength or pH that is manifested in significant changes in the proton NMR spectra of these proteins. Irradiation of the heme 3-CH3 resonances in the proton NMR spectra of tuna, horse and yeast iso-1 ferricytochromes c is shown to give NOE connectivities to the phenyl ring protons of Phe82 as well as to the beta-CH2 protons of this residue. This method was used to probe selectively the Phe82 spin systems of the three cytochromes c under a variety of solution conditions. This phenylalanine residue has previously been shown to be invariant in all mitochondrial cytochromes c, located near the exposed heme edge in proximity to the heme 3-CH3, and may function as a mediator in electron transfer reactions [Louie, G. V., Pielak, G. J., Smith, M. & Brayer, G. D. (1988) Biochemistry 27, 7870-7876]. Ferricytochromes c from all three species undergo a small but specific structural rearrangement in the environment around the heme 3-CH3 group upon changing the solution conditions from low to high ionic strength. This structural change involves a decrease in the distance between the Phe82 beta-CH2 group and the heme 3-CH3 substituent. In addition, studies of the effect of pH on the 1H-NMR spectrum of yeast iso-1 ferricytochrome c show that the heme 3-CH3 proton resonance exhibits a pH-dependent shift with an apparent pK in the range of 6.0-7.0. The chemical shift change of the yeast iso-1 ferricytochrome c heme 3-CH3 resonance is not accompanied by an increase in the linewidth as previously described for horse ferricytochrome c [Burns, P. D. & La Mar, G. N. (1981) J. Biol. Chem. 256, 4934-4939]. These spectral changes are interpreted as arising from an ionization of His33 near the C-terminus. In general, the larger spectral changes observed for the resonances in the vicinity of the heme 3-CH3 group in yeast iso-1 ferricytochrome c with changes in solution conditions, relative to the tuna and horse proteins, suggest that the region around Phe82 is more open and that movement of the Phe82 residue is less constrained in yeast ferricytochrome c. Finally, it is demonstrated here that both the heme 8-CH3 and the 7 alpha-CH resonances of yeast ferricytochrome c titrate with p2H and exhibit apparent pK values of approximately 7.0. The titrating group responsible for these spectral changes is proposed to be His39.     

Cytochrome c reduction by cysteine plus copper: a pseudosubstrate system for cytochrome c oxidase

Cysteine alone reduces horse heart cytochrome c very slowly (k approximately or equal too 1.0 M-1s-1) with a rate constant virtually identical in high and low ionic strength buffers. Copper catalyzes this reaction increasing the rate by a factor of 10(5) in 50 mM phosphate and by a factor of 10(6) in 10mM Tris buffers. When ferricytochrome c and cysteine are mixed in an oxygen electrode a "burst" of oxygen uptake is seen, the decline in which parallels the reduction of cytochrome c. When cytochrome oxidase is added to such a mixture two routes of electron transfer to oxygen exist: enzymatic and ferricytochrome c dependent nonenzymatic. Both processes are sensitive to cyanide, but azide inhibits only the authentic cytochrome c oxidase catalyzed process and BCS the ferricytochrome c stimulated reaction.     

Equilibrium thermodynamics of a physiologically-relevant heme-protein complex

We used isothermal titration calorimetry to study the equilibrium thermodynamics for formation of the physiologically-relevant redox protein complex between yeast ferricytochrome c and yeast ferricytochrome c peroxidase. A 1:1 binding stoichiometry was observed, and the binding free energies agree with results from other techniques. The binding is either enthalpy- or entropy-driven depending on the conditions, and the heat capacity change upon binding is negative. Increasing the ionic strength destabilizes the complex, and both the binding enthalpy and entropy increase. Increasing the temperature stabilizes the complex, indicating a positive van't Hoff binding enthalpy, yet the calorimetric binding enthalpy is negative (-1.4 to -6.2 kcal mol(-)(1)). We suggest that this discrepancy is caused by solvent reorganization in an intermediate state. The measured enthalpy and heat capacity changes are in reasonable agreement with the values estimated from the surface area change upon complex formation. These results are compared to those for formation of the horse ferricytochrome c/yeast ferricytochrome c peroxidase complex. The results suggest that the crystal and solution structures for the yeast complex are the same, while the crystal and solution structures for horse cytochrome c/yeast cytochrome c peroxidase are different.     

Reduction of horse heart ferricytochrome c by glutathione: evidence for two different conformational forms of ferricytochrome c in neutral and slightly alkaline solutions.

The reduction of horse heart ferricytochrome c in neutral and slightly alkaline solutions follows biphasic kinetics. The obtained results are consistent with the existence of two conformational forms of ferricytochrome c, cyt c and cyt c¹. The conversion of cyt c to cyt c¹ occurs by deprotonation (pK～7), followed by a slow conformational change in the protein structure.     

Study of the biological significance of cytochrome methylation. I. Thermal, acid and guanidinium hydrochloride denaturations of baker's yeast ferricytochromes c.

The iso-cytochromes c from baker's yeast: iso-1 methylated and unmethylated forms and iso-2 have been purified and their stabilities towards denaturants compared to that of horse heart cytochrome c. Thermal, acid and guanidinium hydrochloride denaturations were followed using fluorescence emission of their tryptophan 59 and/or the absorbance in the Soret region as the physical parameters. Very few differences could be evidenced among the ferricytochromes investigated in this study insofar as the acid denaturations are concerned. This is to be contrasted with the conclusions of the thermal and guanidinium hydrochloride denaturations studies which clearly showed the ferricytochrome from horse heart to be much more stable than those from baker's yeast. No appreciable differences could be measured among the methylated and unmethylated forms of iso-1 cytochrome c nor among iso-1 and iso-2 cytochromes from baker's yeast. Our results suggest that a stabilizing effect of methylation on the tridimensional structure of ferricytochrome c must probably be discarded. Other possible physiological roles of methylation are suggested taking into account the relative instability of ascomycetes's cytochromes as compared to mammalian ones.     

Evolutionary change of the heme c electronic structure: ferricytochrome c-551 from Pseudomonas aeruginosa and horse heart ferricytochrome c.

Individual assignments of the ¹H n.m.r. lines of heme c in reduced and oxidized cytochrome c-551 from $\text{Pseudomonas aeruginosa}$ were obtained by nuclear Overhauser enhancement and saturation transfer experiments. Comparison with the corresponding data on horse heart cytochrome c showed that the locations of high spin density on the heme c periphery as well as the in-plane principal axes x and y of the electronic g-tensor are rotated by approximately 90° in ferricytochrome c-551 relative to horse ferricytochrome c. High spin density in ferricytochrome c-551 is thus localized on the pyrrole ring III. While this pyrrole ring is well shielded in the interior of mammalian-type cytochromes c, it is more easily accessible in cytochrome c-551. It is suggested that this evolutionary change of the heme c electronic structure would be compatible with the hypothesis that the electron transfer in both species is via solvent exposed peripheral ring carbon atoms.     

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.     

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.     

Alkaline isomerization of thermoresistant cytochrome c-552 and horse heart cytochrome c studied by absorption and resonance Raman spectroscopy.

The structure of the thermoresistant cytochrome c (552, Thermus thermophilus) has been investigated at neutral and alkaline pH by absorption and resonance Raman spectroscopy and compared with that of horse heart cytochrome c. The ligands of the ferricytochrome c-552 at neutral pH are considered to be histidine and methionine, whereas the ligands of ferrocytochrome c-552 are histidine and another nitrogen base, histidine or lysine. Ferric cytochrome c-552 undergoes an alkaline isomerization with a pK of 12.3 (25 degrees C), accompanied by a ligand exchange. Horse heart cytochrome c has at least three isomerization states at alkaline pH (pK 9.3, 12.9 and greater than 13.5 at 25 degrees C). The replacement of the sixth ligand may not be involved in the second isomerization. The thermodynamic parameters for the isomerization were also estimated. The entropy change upon isomerization of cytochrome c-552 is negative, whereas for that of horse heart cytochrome c the entropy change is positive.     

A quantitative test for superoxide radicals produced in biological systems.

The preparation and properties of a partially succinoylated cytochrome c, suited for the detection of superoxide anion radicals in liver microsomes, is reported. By succinoylation of 45% of the primary amino groups of horse heart cytochrome c the activity towards solubilized NADPH--cytochrome P-450 reductase was diminished by 99% compared with native cytochrome c. The capacities of cytochrome b5 and cytochrome c oxidase to reduce the succinoylated ferricytochrome c and oxidize succinoylated ferrocytochrome c respectively were decreased to a similar extent. However, the bimolecular rate constant for the reduction of the partially succinoylated ferricytochrome c by O2-. was estimated to be one-tenth of the value for the reaction of O2-. with native ferricytochrome c a pH 7.7. On this basis the quantification of O2-. generated by NADPH-supplemented liver microsomes became possible. The initial rates of succinoylated ferricytochrome c reduction determined at various finite concentrations of the cytochrome c derivative can be extrapolated to obtain true rates of O2-. generation in a homogeneous system. The problems encountered in the quantitative determination of O2-. produced in biological membranes, e.g. microsomes, are discussed.     

Characterization of pH-dependent conformational heterogeneity in Rhodospirillum rubrum cytochrome c2 using 15N and 1H NMR

The 15N-enriched ferricytochrome c2 from Rhodospirillum rubrum has been studied by 15N and 1H NMR spectroscopy as a function of pH. The 15N resonances of the heme and ligand tau nitrogen are broadened beyond detection because of paramagnetic relaxation. The 15N resonance of the ligand histidine phi nitrogen was unambiguously identified at 184 ppm (pH 5.6). The 15N resonances of the single nonligand histidine are observed only at low pH, as in the ferrocytochrome because of the severe broadening caused by tautomerization. The dependence of the 15N and 1H spectra of the ferricytochrome on pH indicated that the ligand histidine tau NH does not dissociate in the neutral pH range and is involved in a hydrogen bond, similar to that in the reduced state. Because neither deprotonated nor non-hydrogen-bonded forms of the ligand histidine are observed in the spectra of either oxidation state, the participation of such forms in producing heterogeneous populations having different electronic g tensors is ruled out. Transitions having pKa's of 6.2, 8.6, and 9.2 are observed in the ferricytochrome. The localized conformational change around the omega loops is observed in the neutral pH range, as in the ferrocytochrome. Structural heterogeneity leads to multiple resonances of the heme ring methyl at position 8. The exchange rate between the conformations is temperature dependent. The transition with a pKa of 6.2 is assigned to the His-42 imidazole group. The displacement of the ligand methionine, which occurs with a pKa of 9.2, causes gross conformational change near the heme center.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

The solution structures of tuna and horse cytochromes c

The nuclear magnetic resonance spectra of tuna ferricytochrome c and tuna ferrocytochrome c are described. Resonance assignments are made using NMR double-resonance techniques. A comparison of the NMR data for tuna cytochrome c with the previously reported data for horse cytochrome c shows that the proteins have virtually identical main-chain folds. Three regions of local conformational differences have been distinguished.