Physicochemical characterization of the alkaline denaturation of cytochrome c peroxidase

The alkaline denaturation of cytochrome c peroxidase and apocytochrome c peroxidase was investigated by analytical ultracentrifugation, gel-filtration chromatography, and circular dichroism. The results indicate that both cytochrome c peroxidase and the apoenzyme undergo extensive structural modifications upon exposure to alkaline pH, including dimer formation. The midpoint of the transition for dimer formation in the native enzyme occurs at pH 9.6 +/- 0.1, while loss of tertiary and secondary structure occurs with transition midpoints at pH 10.3 +/- 0.1 and pH 11.3 +/- 0.1, respectively. Studies performed in the presence of dithiothreitol and with carboxymethylated cytochrome c peroxidase indicate that dimer formation occurs via a disulfide crosslink involving the single cysteine residue in the enzyme. Denaturation of cytochrome c peroxidase in the presence of guanidine hydrochloride gave results similar to those obtained for the alkaline denaturation.     

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.     

Characterization of horse cytochrome c expressed in Escherichia coli.

We have expressed horse cytochrome c in Escherichia coli. The gene was designed with E. coli codon bias and assembled by using a recursive polymerase chain reaction method. The far-ultraviolet and near-ultraviolet/Soret circular dichroism (CD) spectra show that the structure of recombinant horse cytochrome c is the same as that of the authentic protein. CD-detected thermal denaturation studies were used to measure the thermodynamic parameters associated with two-state denaturation. The free energy of denaturation for the recombinant protein is 10.0 +/- 2.3 kcal mol(-1) at pH 4.6 and 25 degrees C, which agrees with the value for the authentic protein. The expression system will help advance our understanding of the roles of cytochrome c in electron transfer, oxidative stress, and apoptosis by allowing the production of protein variants.     

Membrane binding induces destabilization of cytochrome c structure.

The effect of membranes binding on the structure and stability of ferricytochrome c was studied by Fourier-transform infrared spectroscopy and differential scanning calorimetry. Association of cytochrome c with phospholipid membranes containing phosphatidylglycerol as a model acidic phospholipid results in only slight, if any, perturbation of the protein secondary structure. However, upon membrane binding, there is a considerable increase in the accessibility of protein backbone amide groups to hydrogen-deuterium exchange, which suggests a lipid-mediated loosening and/or destabilization of the protein tertiary structure. A lipid-induced conformational perturbation of ferricytochrome c is also indicated by a marked decrease in the thermodynamic stability of the membrane-bound protein. Upon binding to membranes containing dimyristoylphosphatidylglycerol (DMPG) or dioleoylphosphatidylglycerol (DOPG) as a single lipid component, the denaturation temperature of ferricytochrome c decreases by approximately 30 degrees C. This is accompanied by a decrease in the calorimetric enthalpy of denaturation, particularly for the DMPG-associated protein. With ferricytochrome c bound to membranes containing a mixture of DMPG (or DOPG) and zwitterionic phosphatidylcholine, the extent of structural perturbation depends on the surface density of the negatively charged lipid head groups, becoming smaller with decreasing proportions of acidic phospholipid in the membrane. The observed destabilization of protein structure mediated by acidic phospholipids (and possibly formation of folding intermediates at the membrane surface) may represent a general property of a larger class of water-soluble proteins for which membrane binding is governed by electrostatic forces.     

Stabilization of Pseudomonas aeruginosa cytochrome c(551) by systematic amino acid substitutions based on the structure of thermophilic Hydrogenobacter thermophilus cytochrome c(552)

A heterologous overexpression system for mesophilic Pseudomonas aeruginosa holocytochrome c(551) (PA c(551)) was established using Escherichia coli as a host organism. Amino acid residues were systematically substituted in three regions of PA c(551) with the corresponding residues from thermophilic Hydrogenobacter thermophilus cytochrome c(552) (HT c(552)), which has similar main chain folding to PA c(551), but is more stable to heat. Thermodynamic properties of PA c(551) with one of three single mutations (Phe-7 to Ala, Phe-34 to Tyr, or Val-78 to Ile) showed that these mutants had increased thermostability compared with that of the wild-type. Ala-7 and Ile-78 may contribute to the thermostability by tighter hydrophobic packing, which is indicated by the three dimensional structure comparison of PA c(551) with HT c(552). In the Phe-34 to Tyr mutant, the hydroxyl group of the Tyr residue and the guanidyl base of Arg-47 formed a hydrogen bond, which did not exist between the corresponding residues in HT c(552). We also found that stability of mutant proteins to denaturation by guanidine hydrochloride correlated with that against the thermal denaturation. These results and others described here suggest that significant stabilization of PA c(551) can be achieved through a few amino acid substitutions determined by molecular modeling with reference to the structure of HT c(552). The higher stability of HT c(552) may in part be attributed to some of these substitutions.     

Spectral studies of horse heart porphyrin cytochrome c

Removal of the heme iron from cytochrome c to generate porphyrin cytochrome c relieves the quenching of porphyrin fluorescence and enhances the fluorescence of the single tryptophan residue and the 4 tyrosine residues. The intensity of the porphyrin fluorescence is not perturbed by denaturation of the protein at neutral pH using either urea or guanidine hydrochloride. However, the amplitude of tryptophan fluorescence is increased by these denaturants from 5 to about 85% of a model tryptophan residue using solutions of 2 microM tryptophan. In contrast to cytochrome c, the tryptophan fluorescence amplitude of denatured porphyrin cytochrome c is independent of pH over the range pH 3.0 to 7.4. Acidification of solutions of either native or denatured porphyrin cytochrome c markedly alters both the visible absorbance and fluorescence of the protein consistent with protonation of two pyrrole nitrogens on the porphyrin. Preliminary analysis of the spectral changes occurring in the acid transition suggests the presence of an intermediate form having only one of these two pyrrole nitrogens protonated.     

Thermal properties of chemically modified cytochrome c

Differential scanning calorimetry was used as a probe to follow structural disturbances in cytochrome c with electrostatic modification. At 51.7% maleylation, the Td decreased 14.1 degrees C; however, relatively stable delta H values reflected minor structural variations. With 77.5 and 96.4% modification, a significant decrease in delta H was more indicative of major conformational change. On this basis, a critical labelling point was considered. Extensive maleylation (96.4%) did not result in complete cytochrome denaturation. In general, assessment of cytochrome thermal parameters by DSC provided a conformational perspective for the influence of specific electrostatic parameters on molecular integrity.     

Thermal stability of membrane-reconstituted yeast cytochrome c oxidase

The thermal dependence of the structural stability of membrane-reconstituted yeast cytochrome c oxidase has been studied by using different techniques including high-sensitivity differential scanning calorimetry, differential detergent solubility thermal gel analysis, and enzyme activity measurements. For these studies, the enzyme has been reconstituted into dimyristoylphosphatidylcholine (DMPC) and dielaidoylphosphatidylcholine (DEPC) vesicles using detergent dialysis. The phospholipid moiety affects the stability of the enzyme as judged by the dependence of the denaturation temperature on the lipid composition of the bilayer. The enzyme is more stable when reconstituted with the 18-carbon, unsaturated phospholipid (DEPC) than with the 14-carbon saturated phospholipid (DMPC). In addition, the shapes of the calorimetric transition profiles are different in the two lipid systems, indicating that not all of the subunits are affected equally by the lipid moiety. The overall enthalpy change for the enzyme denaturation is essentially the same for the two lipid reconstitutions (405 kcal/mol of protein for the DMPC and 425 kcal/mol for the DEPC-reconstituted enzyme). In both systems, the van't Hoff to calorimetric enthalpy ratios are less than 0.2, indicating that the unfolding of the enzyme cannot be represented as a two-state process. Differential detergent solubility experiments have allowed us to determine individual subunit thermal denaturation profiles. These experiments indicate that the major contributors to the main transition peak observed calorimetrically are subunits I and II and that the transition temperature of subunit III is the most affected by the phospholipid moiety. Experiments performed at different scanning rates indicate that the thermal denaturation of the enzyme is a kinetically controlled process characterized by activation energies on the order of 40 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Resolution of bovine heart cytochrome c oxidase into smaller complexes by controlled subunit denaturation

Bovine heart cytochrome c oxidase has been partially denaturated under mild conditions with 0.1-0.25% lithium dodecyl sulfate and 0.05% Triton X-100. From its reactivity towards CO and CN-, an unmasking of the heme a was inferred in this enzyme. The catalytic activity was lost during the denaturation and small spectral differences became visible. Spectra and ligand binding properties of the denatured enzyme were reversed by dilution in 2% Triton X-100. This suggests that during the denaturation procedure the hemes were not displaced from their original sites. By gel filtration of the partially denatured enzyme the following complexes of subunits were obtained: I-III, I-II-III, II-IV-V-VI-VII and IV-V-VI-VII. The first three complexes retained almost all the heme, and their spectral characteristics were very similar to those of the partially denatured cytochrome c oxidase. The data, in combination with the information that subunit III does not contain heme [Saraste et al. (1980) FEBS Lett. 114, 35-38], suggest that the hemes are attached to subunit I and II. After denaturation of cytochrome c oxidase under more drastic conditions some of the heme was also found to be associated with the smaller subunits, but its spectral characteristics were radically altered, becoming almost identical to those of free heme.     

Folding of yeast iso-1-AM cytochrome c

We describe a specific modification of iso-1 cytochrome c which results in blocking a single free sulfhydryl group. The derivative differs from the unmodified protein by the introduction of a small, uncharged group, thus maintaining the same charge balance as the native protein. The modified protein, obtained by treatment of iso-1 cytochrome c with iodoacetamide, has an activity indistinguishable from that of the unmodified protein in the lactate dehydrogenase-cytochrome c reductase system from yeast and has the same stability toward denaturation by guanidine hydrochloride. The kinetics of fluorescence changes associated with the guanidine hydrochloride induced folding-unfolding transition for modified iso-1 cytochrome c (iso-1-AM) have been investigated throughout the transition zone by using stopped-flow mixing. The results are compared to those for the yeast isozyme, iso-2 cytochrome c. The main features of the fluorescence-detected folding kinetics are similar, as might be expected for homologous proteins; however, the limiting value of the fraction of fast refolding protein (alpha 2) below the transition zone is smaller for iso-1-AM (approximately 0.7) than for iso-2 (approximately 0.9).     

Calorimetric studies of the thermal denaturation of cytochrome c peroxidase

Two endotherms are observed by differential scanning calorimetry during the thermal denaturation of cytochrome c peroxidase at pH 7.0. The transition midpoint temperatures (tm) were 43.9 +/- 1.4 and 63.3 +/- 1.6 degrees C, independent of concentration. The two endotherms were observed at all pH values between 4 and 8, with the transition temperatures varying with pH. Precipitation was observed between pH 4 and 6, and only qualitative data are presented for this region. The thermal unfolding of cytochrome c peroxidase was sensitive to the presence and ligation state of the heme. Only a single endotherm was observed for the unfolding of the apoprotein, and this transition was similar to the high-temperature transition in the holoenzyme. Addition of KCN to the holoenzyme increases the midpoint of the high-temperature transition whereas the low-temperature transition was increased upon addition of KF. Binding of the natural substrate ferricytochrome c to the enzyme increases the low-temperature transition by 4.8 +/- 1.3 degrees C but has no effect on the high-temperature transition at pH 7. The presence of cytochrome c peroxidase decreases the stability of cytochrome c, and both proteins appear to unfold simultaneously. The results are discussed in terms of the two domains evident in the X-ray crystallographic structure of cytochrome c peroxidase.     

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.     

Appearance of nitrite reducing activity of cytochrome c upon heat denaturation

The appearance of NO2- reducing activity of cytochrome c (Cyt c) upon heat denaturation was investigated with equine heart Cyt c. Denatured equine heart Cyt c (dCyt c), which was treated at 100 degrees C for 30 min, had NO2- reducing activity in the presence of dithionite and methylviologen in an aqueous solution under anaerobic conditions. In contrast, hemoglobin and myoglobin had no such activity under the same conditions. Using spectroscopic methods, we found that the appearance of this activity in the Cyt c was due to the following intramolecular changes: unfolding of the peptide chain, exposure of the heme, dissociation of the sixth ligand methionine sulfur, and appearance of autoxidizability. The dCyt c catalyzed NO2- reduction to NH4+ via ferrous-NO complexes, and this reaction was a 6-electron and 8-proton reduction. Sepharose-immobilized dCyt c had activity similar strength to that in solution. The resin retained the activity after five uses and even after storage for 1 year. On the basis of these results, we concluded that Cyt c acquired a new catalytic activity upon heat treatment, unlike to other familiar biological molecules.     

Optical spectroscopic differentiation of various equilibrium denatured states of horse cytochrome c

Thermal unfolding of cytochrome c (cyt c) from several states has been studied using equilibrium spectroscopic techniques. CD in the uv, vibrational circular dichroism, infrared, and uv-vis absorption spectra measured at various temperatures, pHs, salt concentrations, and GuHCl concentrations are used to show the conformational as well as heme structural differences between native and various denatured states. The difference in thermal denaturation behaviors of cyt c starting from acid denatured, molten globule (MG), and the A and native states are explored. Different final high temperature states were observed for cytochrome c unfolding from four different initial states (native, MG, A, and acid denatured state) by electronic CD, Fourier transform infrared (FTIR), and vibrational CD (VCD). Consistent with this, different thermal unfolding pathways for the MG and A states are suggested by the FTIR and VCD data for this process.     

Introduction of a disulfide bond into cytochrome c stabilizes a compact denatured state.

We introduced a novel disulfide bond, modeled on that of bullfrog cytochrome c, into yeast iso-1-cytochrome c. The disulfide spontaneously forms upon purification. A variety of techniques were used to examine the denaturation of this variant and several non-cross-linked controls. Denaturation is reversible and, with the exception of the protein in which the two cysteines are blocked, consistent with a two-state process. Comparison of the calorimetric and van't Hoff enthalpy changes indicates that denaturation is two-state at pH 4.6. Calorimetric and fluorescence-monitored guanidine hydrochloride (GdnHCl) denaturation data indicate that the free energy of denaturation for the cross-linked protein (delta Gd at 300 K) is decreased relative to non-cross-linked controls. The dependence of delta Gd on GdnHCl concentration, the GdnHCl concentration that denatures half the protein, as well as the enthalpy, entropy, and heat capacity changes (mGdnHCl, Cm, delta Hd, delta Sd, and delta Cp, respectively), all decrease in magnitude upon introduction of the cross-link. The decrease in delta Hd and delta Sd were confirmed by monitoring absorbance at several wavelengths as a function of temperature. The cross-link also decreases the pH dependence of these observables. Circular dichroism studies indicate the denatured state of the cross-linked protein possesses more structure than non-cross-linked proteins, and this structure is refractory to increases in temperature and chemical denaturant. We conclude that the diminished values of delta Gd, delta Hd, delta Sd, delta Cp, and mGdnHCl result from the denatured state of the cross-linked variant being more compact and possessing more structure than non-cross-linked controls.     

Anion and ionic strength effects upon the oxidation of cytochrome c by cytochrome c oxidase

Citrate and other polyanion binding to ferricytochrome c partially blocks reduction by ascorbate, but at constant ionic strength the citrate-cytochrome c complex remains reducible; reduction by TMPD is unaffected. At a constant high ionic strength citrate inhibits the cytochrome c oxidase reaction competitively with respect to cytochrome c, indicating that ferrocytochrome c also binds citrate, and that the citrate-ferrocytochrome c complex is rejected by the binding site at high ionic strength. At lower ionic strengths, citrate and other polyanions change the kinetic pattern of ferrocytochrome c oxidation from first-order towards zero-order, indicating preferential binding of the ferric species, followed by its exclusion from the binding site. The turnover at low cytochrome c concentrations is diminished by citrate but not the Km (apparent non-competitive inhibition) or the rate of cytochrome a reduction by bound cytochrome c. Small effects of anions are seen in direct measurements of binding to the primary site on the enzyme, and larger effects upon secondary site binding. It is concluded that anion-cytochrome c complexes may be catalytically competent but that the redox potentials and/or intramolecular behaviour of such complexes may be affected when enzyme-bound. Increasing ionic strength diminishes cytochrome c binding not only by decreasing the 'association' rate but also by increasing the 'dissociation' rate for bound cytochrome c converting the 'primary' (T) site at high salt concentrations into a site similar kinetically to the 'secondary' (L) site at low ionic strength. A finite Km of 170 microM at very high ionic strength indicates a ratio of K infinity m/K 0 M of about 5000. It is proposed that anions either modify the E10 of cytochrome C bound at the primary (T) site of that they perturb an equilibrium between two forms of bound c in favour of a less active form.     

The dipole moment of cytochrome c.

Vertebrate cytochromes c and the cytochromes c of insects and plants have, on average, dipole moments of 320 and 340 debye, respectively. The direction of the dipole vector with respect to the haem plane, at the solvent-accessible edge of which electron transfer presumably takes place, is conserved in these two groups--at 32 degrees +/- 7 degrees and 22 degrees +/- 10 degrees, respectively. The variation of dipole orientations and magnitudes observed in these species is compared with the results of a model in which charge distributions occur randomly. Since this model does not generate the observed charge asymmetries of the various cytochromes c, it is concluded that the dipole moment of cytochrome c is a feature that is evolutionarily conserved, apparently because it has an important influence on the interaction of this mobile electron carrier with its physiological electron donors and acceptors in the intermembrane space of mitochondria.     

Sequence and stability of the goat cytochrome c

We have determined the sequence of mitochondrial cytochrome c (cyt-c) from the goat heart, and it was found to have a unique amino acid sequence among all amino acid sequences of cyt-c reported till date. Its sequence alignment with the bovine cytochrome c (b-cyt-c) led us to conclude that the goat cytochrome c (g-cyt-c) differs in amino acid sequence from b-cyt-c at only one position, i.e., Pro44(bovine) --> Ala44(goat). It has been observed that guanidinium chloride (GdmCl) induces a two-state transition between the native (N) and denatured (D) states of g-cyt-c. This conclusion is reached from the coincidence of GdmCl-induced transition curves monitored by measurements of absorbance at 405, 530 and 695 nm and circular dichroism (CD) at 222, 416 and 405 nm. Analysis of denaturation curves for the Gibbs energy of stabilization suggests that the stability of g-cyt-c is, within experimental errors, identical to that of b-cyt-c. We have also measured the effect of temperature on the equilibrium, N state <--> D state of g-cyt-c in the presence of different GdmCl concentrations. These measurements gave values of transition temperature (T(m)), changes in enthalpy (DeltaH(m)) and heat capacity (DeltaC(p)) of g-cyt-c in the absence of GdmCl, which are compared with those of b-cyt-c. We have used crystal structure coordinates of b-cyt-c to predict the structure and stability of g-cyt-c, which are compared with those of the bovine protein.     

The cytochrome c peroxidase of Paracoccus denitrificans.

The size, visible absorption spectra, nature of haem and haem content suggest that the cytochrome c peroxidase of Paracoccus denitrificans is related to that of Pseudomonas aeruginosa. However, the Paracoccus enzyme shows a preference for cytochrome c donors with a positively charged 'front surface' and in this respect resembles the cytochrome c peroxidase from Saccharomyces cerevisiae. Paracoccus cytochrome c-550 is the best electron donor tested and, in spite of an acidic isoelectric point, has a markedly asymmetric charge distribution with a strongly positive 'front face'. Mitochondrial cytochromes c have a much less pronounced charge asymmetry but are basic overall. This difference between cytochrome c-550 and mitochondrial cytochrome c may reflect subtle differences in their electron transport roles. A dendrogram of cytochrome c1 sequences shows that Rhodopseudomonas viridis is a closer relative of mitochondria than is Pa. denitrificans. Perhaps a mitochondrial-type cytochrome c peroxidase may be found in such an organism.