Steady state kinetics and binding of eukaryotic cytochromes c with yeast cytochrome c peroxidase.

1. The steady state kinetics for the oxidation of ferrocytochrome c by yeast cytochrome c peroxidase are biphasic under most conditions. The same biphasic kinetics were observed for yeast iso-1, yeast iso-2, horse, tuna, and cicada cytochromes c. On changing ionic strength, buffer anions, and pH, the apparent Km values for the initial phase (Km1) varied relatively little while the corresponding apparent maximal velocities varied over a much larger range. 2. The highest apparent Vmax1 for horse cytochrome c is attained at relatively low pH (congruent to 6.0) and low ionic strength (congruent to 0.05), while maximal activity for the yeast protein is at higher pH (congruent to 7.0) and higher ionic strength (congruent to 0.2), with some variations depending on the nature of the buffering ions. 3. Direct binding studies showed that cytochrome c binds to two sites on the peroxidase, under conditions that give biphasic kinetics. Under those ionic conditions that yield monophasic kinetics, binding occurred at only one site. At the optimal buffer concentrations for both yeast and horse cytochromes c, the KD1 and KD2 values approximate the Km1 and Km2 values. At ionic strengths below optimal, binding becomes too strong and above optimal, too weak. 4. Under ionic conditions that are optimal and give monophasic kinetics with horse cytochrome c but are suboptimal for the yeast protein, yeast cytochrome c strongly inhibits the reaction of horse cytochrome c with peroxidase, uncompetitively at one site and competitively at a second site. The appearance of the second site under monophasic conditions is interpreted as an allosteric effect of the inhibitor binding to the first site. 5. The simplest model accounting for these observations postulates two kinetically active sites on each molecule of peroxidase, a high affinity and a low affinity site, that may correspond to the free radical and the heme iron (IV) of the oxidized enzyme, respectively. Both oxidizing equivalents may be discharged at either site. Furthermore, the enzyme appears to exist as an equilibrium mixture of a high ionic strength form, EH and a low ionic strength form, EL, the former reacting optimally with yeast cytochrome c, and the latter with horse cytochrome c.     

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.     

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.     

Low-temperature studies of electron transfer between different cytochromes c and cytochrome c oxidase.

The ability of various native and modified cytochromes c to transfer electrons to cytochrome oxidase is compared in cytochrome c depleted beef heart mitochondrial particles. The kinetics are followed at -49 degrees C after the reaction is initiated by photolysis of the CO compound of cytochrome oxidase in the presence of oxygen. Horse, human, yeast iso-2, and carboxydinitrophenyl (CDNP)-lysine-60 horse cytochromes c all give initial rates of electron transfer that are equal to those observed in whole beef mitochondria. Euglena, CDNP-lysine-72, and CDNP-lysine-13 horse cytochromes c give rates about one-tenth that of whole mitochondria. These rates were independent of the concentration of cytochrome c. Since the inhibited cytochromes c, but not the active proteins, had previously been shown to have lowered affinity for cytochrome oxidase, the results indicate that the structural characteristics important for the association of cytochrome c and oxidase are also essential for achieving normal rates of electron transfer within the complex once formed.     

Formation of a cytochrome c-like species from horse apoprotein and hemin catalyzed by yeast mitochondrial cytochrome c synthetase

Cytochrome c synthetase in yeast mitochondria catalyzes the formation of a yeast cytochrome c-like species from the apoprotein and hemin (Basile, G., DiBello, C., and Taniuchi, H. (1980) J. Biol. Chem. 255, 7181-7191). To test the specificity of this enzyme, 125I-labeled horse apocytochrome c was incubated with the yeast mitochondrial fraction in the presence of hemin, NADPH, and an ethanol extract of the postmitochondrial fraction. A radioactive 125I-labeled cytochrome c-like species was formed in yields of up to 26%. This 125I-labeled species is indistinguishable from horse cytochrome c by ion exchange chromatography (under the conditions which allow separation of horse and yeast cytochrome c), resistance in its reduced form to digestion by trypsin, resistance against autoxidation, reduction by cytochrome b2, and generation of the apoprotein after treatment with silver sulfate and dithiothreitol. With unlabeled horse apoprotein and [59Fe]hemin, the yield of a [59Fe-labeled horse cytochrome c-like species was up to 7% with respect to the apoprotein incubated. The yield of the 59Fe-labeled species was not altered by the addition of unlabeled FeCl3. Conversely, synthesis of the 59Fe-labeled species was not detectable after incubation of yeast mitochondria with unlabeled horse apoprotein, unlabeled hemin, and 59FeCl3. The formation of both 125I- and 59Fe-labeled cytochrome c-like species was sensitive to heat. Thus, we conclude that cytochrome c synthetase catalyzes direct bonding of heme (or hemin) to the apoprotein. Since the amino acid sequences of horse and yeast cytochromes c differ considerably, cytochrome c synthetase may recognize only a limited region(s) of the apoprotein.     

Stereochemistry of the porphyrin-protein bond of cytochrome c. Stereochemical comparison of Rhodospirillum rubrum, yeast, and horse heart porphyrins c.

Porphyrins c have been obtained from Rhodospirillum rubrum cytochrome c2, yeast cytochrome c, and horse heart cytochrome c and compared using proton magnetic resonance and circular dichroism. Identity of the spectra establishes that chemically and stereochemically the three porphyrins c are identical. Since the stereochemistry of the porphyrin alpha-thioether linkage is not affected in the conversion to porphyrin c, the stereochemistry at the porphyrin alpha-thioether bonds among the corresponding cytochromes c also must be the same. Differences between the proton magnetic resonance of R. rubrum cytochrome c2 and horse heart cytochrome c which were rationalized by invoking an opposite stereochemistry at these condensation sites (Smith, G. M., and Kamen, M. D. (1974), Proc. Natl. Acad. Sci. U.S.A. 71, 4303) must therefore be attributed to other factors.     

Membrane location of apocytochrome c and cytochrome c determined from lipid-protein spin exchange interactions by continuous wave saturation electron spin resonance.

Apocytochrome c derived from horse heart cytochrome c was spin-labeled on the cysteine residue at position 14 or 17 in the N-terminal region of the primary sequence, and cytochrome c from yeast was spin-labeled on the single cysteine residue at sequence position 102 in the C-terminal region. The spin-labeled apocytochrome c and cytochrome c were bound to fluid bilayers composed of different negatively charged phospholipids that also contained phospholipid probes that were spin-labeled either in the headgroup or at different positions in the sn-2 acyl chain. The location of the spin-labeled cysteine residues on the lipid-bound proteins was determined relative to the spin-label positions in the different spin-labeled phospholipids by the influence of spin-spin interactions on the microwave saturation properties of the spin-label electron spin resonance spectra. The enhanced spin relaxation observed in the doubly labeled systems arises from Heisenberg spin exchange, which is determined by the accessibility of the spin-label group on the protein to that on the lipid. It is found that the labeled cysteine groups in horse heart apocytochrome c are located closest to the 14-C atom of the lipid acyl chain when the protein is bound to dimyristoyl- or dioleoyl-phosphatidylglycerol, and to that of the 5-C atom when the protein is bound to a dimyristoylphosphatidylglycerol/dimyristoylphosphatidylcholine (15:85 mol/mol mixture. On binding to dioleoylphosphatidylglycerol, the labeled cysteine residue in yeast cytochrome c is located closest to the phospholipid headgroups but possibly between the polar group region and the 5-C atom of the acyl chains. These data determine the extent to which the different regions of the proteins are able to penetrate negatively charged phospholipid bilayers.     

On the specificity of cytochrome c synthetase in recognition of the amino acid sequence of apocytochrome c

Two forms of yeast cytochrome c synthetases with different specificities were resolved, one (synthetase I), solubilized from mitochondria or the cell debris with Triton X-100, recognizing not horse apocytochrome c but yeast apo-iso-1-cytochrome c as a substrate and the other (synthetase II) still bound with the particulate fraction from mitochondria after treatment with Triton, recognizing both horse and yeast apocytochromes c. The activity with labeled yeast apo-iso-1-cytochrome c as a substrate of cytochrome c synthetase I can be quantitatively inhibited by nonlabeled Candida krusei apocytochrome c and partially by nonlabeled tuna apocytochrome c but not by nonlabeled horse apocytochrome c indicating a specific amino acid sequence being recognized. However, an enzyme similarly solubilized from beef heart mitochondria recognized both horse apocytochrome c and yeast apo-iso-1-cytochrome c for attachment of heme. In view of the fact that the yeast synthetase II and the beef synthetase can both utilize either horse apocytochrome c or yeast apo-iso-1-cytochrome c as substrates, we suggest that these enzymes may also be involved in biosynthesis of cytochrome c1, that is, the ability to attach heme to apocytochrome c and apocytochrome c1 may have been conserved in eucaryotic cells, and that both synthetases may therefore be homologous.     

Cytochromes c of Nitrobacter winogradskyi and Thiobacillus novellus: structure, function and evolution.

The amino acid sequences of Thiobacillus novellus and Nitrobacter winogradskyi cytochromes c have been compared with those of cytochromes c from several other organisms. The two bacterial cytochromes resemble eukaryotic cytochromes c; 49 amino-acid residues are identical between T. novellus and horse cytochromes c, and 50 residues identical between N. winogradskyi and horse cytochromes c. However, their reactivity with cow cytochrome c oxidase is about 80% lower than the reactivity of eukaryotic cytochromes c with the cow mitochondrial oxidase, while they react with yeast cytochrome c peroxidase as rapidly as eukaryotic cytochromes c. The numbers of identical amino-acid residues between T. novellus and animal cytochromes c are 45-53 and those between N. winogradskyi and animal cytochromes c 47-53, while those between the two bacterial cytochromes and yeast and protozoan cytochromes c are around 40. Thus, N. winogradskyi and T. novellus cytochromes c are more similar to animal cytochromes c than to yeast and protozoan cytochromes c on the basis of the amino-acid sequence.     

Pulse radiolysis study of a yeast cytochrome c from Hansenula anomala

The reduction of Hansenula anomala yeast cytochrome c by e-aq and CO-.2 was investigated by pulse radiolysis, at a high reductant to protein concentration ratio. The reactivity of the radicals was studied by observing absorbance changes in the cytochrome c spectrum over the wavelength range 280-600 nm. At pH 7, over the time scale of the radical decays (i.e. 0-4 microseconds for e-aq; 0-40 microseconds for CO-.2s) and beyond, the hemoprotein was reduced without any spectrally detected intermediate between ferri-and ferro-forms. This conclusion was reached by simulation studies based on the direct reduction of the yeast cytochrome c from the ferri- to the ferro-form, yielding a correct fit between experimental and calculated absorbance curves. The reduction rate constants were determined to be 1.0 +/- 01 X 10(10) M-1 S-1 for e-aq and 0.7 +/- 0.05 X 10(9) M-1 S-1 for CO-.2 at 0.16 M ionic strength, pH 7.0 and 20 degrees C, thus not significantly different from other values reported for horse heart cytochrome c. However, in the 360-390 nm region the generation of an additional radical species was noticed. The present experimental data were compared with previously published reports.     

Iso-cytochrome c species from baker''s yeast. Analysis of their circular-dichroism spectra.

The circular-dichroism spectra of baker's-yeast iso-1- (methylated and unmethylated forms) and iso-2-cytochrome c species were examined between 200 and 600nm. In the visible region the yeast haemoproteins have characteristics nearly indistinguishable from those of horse heart cytochrome c. From the spectra in the u.v. region the latter appears, however, to be more helical. It is proposed that the likely element of non-helical structure in iso-1-cytochrome c is residues 62-70.     

Alkaline isomerization of horse and yeast cytochromes C. Spectrophotometric and circular dichroism studies.

Spectrophotometric studies of the alkaline isomerization of horse heart and yeast cytochrome c show that the haemoproteins from Saccharomyces cerevisiae differ significantly from the mammalian cytochrome c. Apparent pKa values of 8.41, 8.40 and 8.73 for isol-1-(the methylated and unmethylated forms) and iso-2-cytochrome c respectively, from baker's yeast were determined and compared with the value of 9.40 found for horse heart cytochrome c. The transitions, measured by observing the decrease of the absorbance at 695 nm as the pH increases, have been found to strictly parallel the decrease in amplitude of the negative circular dichroism band centered at 417 nm. This observation gives additional evidence that this negative band is closely related to the ligation of the heme iron by the sulfur atom of methionine 8u for each of the four haemoproteins examined.     

Kinetic studies on redox reactions of hemoproteins. I. Reduction of thermoresistant cytochrome c-552 and horse heart cytochrome c by ferrocyanide.

The oxidation-reduction reaction of horse heart cytochrome c and cytochrome c (552, Thermus thermophilus), which is highly thermoresistant, was studied by temperature-jump method. Ferrohexacyanide was used as reductant. (Formula: see text.) Thermodynamic and activation parameters of the reaction obtained for both cytochromes were compared with each other. The results of this showed that (1) the redox potential of cytochrome c-552, + 0.19 V, is markedly less than that of horse heart cytochrome c. (2) deltaHox of cytochrome c-552 is considerably lower than that of horse heart cytochrome c. (3) deltaSox and deltaSred of cytochrome c-552 are more negative than those of horse heart cytochrome c. (4) kred of cytochrome c-552 is much lower than that of horse heart cytochrome c at room temperature.     

Protein--protein docking of electron transfer complexes: cytochrome c oxidase and cytochrome c

Electron transferring protein complexes form only transiently and the crystal structures of electron transfer protein--protein complexes involving cytochrome c could so far be determined only for the pairs of yeast cytochrome c peroxidase (CcP) with iso-1-cytochrome c (iso-1-cyt c) and with horse heart cytochrome c (cyt c). This article presents models from computational docking for complexes of cytochrome c oxidase (COX) from Paracoccus denitrificans with horse heart cytochrome c, and with its physiological counterpart cytochrome c552 (c552). Initial docking is performed with the FTDOCK program, which permits an exhaustive search of translational and rotational space. A filtering procedure is then applied to reduce the number of complexes to a manageable number. In a final step of structural and energetic refinement, the complexes are optimized by rigid-body energy minimization with the molecular mechanics package CHARMM. This methodology was first tested on the CcP:iso-1-cyt c complex, in which the complex with the lowest CHARMM energy has an RMSD from the crystal structure of only 1.8 A (C(alpha) carbon atoms). Notably, the crystal conformation has an even lower energy. The same procedure was then applied to COX:cyt c and COX:c552. The lowest-energy COX:cyt c complex is very similar to a docking model previously described for the complex of bovine cytochrome c oxidase with horse heart cytochrome c. For the COX:c552 complex, cytochrome c552 is found in two different orientations, depending on whether it is docked against COX from a two-subunit or from a four-subunit crystal structure, respectively. Both conformations are discussed critically in the light of the available experimental data.     

Topographic antigenic determinants on cytochrome c. Immunoadsorbent separation of the rabbit antibody populations directed against horse cytochrome.

Seven populations of site-specific antibodies were isolated from each of three sera of rabbits immunized against glutaraldehyde-polymerized horse cytochrome c. The antibodies were separated using an immunoadsorption scheme which employed the following cytochromes c: horse, beef, guanaco, rabbit, mouse testicular, pigeon, and the cyanogen-bromide cleaved fragment of the rabbit protein containing residues 1 to 65. The monovalent, antigen-binding fragments of the antibodies (Fab') gave 1:1 stoichiometries with native horse cytochrome c in fluorescence quenching assays. Cross-reactivities with heterologous cytochromes c using fluorescence quenching and a modified Farr assay demonstrated that the antigenic determinants are situated around residues 44, 60, and 89/92, four of the six amino acid sequence positions where horse and rabbit cytochromes c differ. The remaining two differences occur at residues 47 and 62. The apparent lack of immunogenicity of these two substitutions may result from the presence of the more immunogenic residues 44 and 60 nearby. Of the seven antibody populations isolated, four were shown to bind in the region of residues 89 and 92. Since several cytochromes c have amino acid sequence differences from the horse protein at either of these two residue positions, it was possible to fractionate the antibodies directed against this complex site on the basis of subtle specificity differences between them. Two antibody populations bind in the region of residue 44. One of these is specific for proline at that position, while the other antibody population also binds to cytochrome c containing glutamic acid at position 44. The remaining antibody population binds in the region of the lysine residue at position 60. Each of the seven site-specific antibody populations binds effectively to any cytochrome c having a suitable amino acid sequence in the antigenic determinant regardless of any residue differences from the immunogen outside of that area. It was also demonstrated that these seven antibody populations represent the totality of the antibodies elicited in rabbits against horse cytochrome c, since the immunoadsorbants bound all the antibodies specific for the native protein. Furthermore, the rabbit antisera contained no other antibody population that could bind to the conformationally disturbed, cyanogen bromide-cleaved fragment of horse cytochrome c containing residues 1 to 65, making it appear that there were no antibodies elicited against a "processed" form of cytochrome c.     

The primary structure of cytochrome c from the rust fungus Ustilago sphaerogena

1. The complete amino acid sequence of cytochrome c from the basidiomycete Ustilago sphaerogena was determined from the amino acid compositions and sequences of either tryptic or chymotryptic peptides, and in homology with at least thirty other established sequences of cytochrome c. 2. The primary structure of the molecule bears all of the characteristics of a mammalian-type cytochrome c, showing the typical clustered distribution of hydrophobic and basic residues with a single polypeptide chain of 107 residues. 3. Like all other fungal cytochromes c, it possesses a free N-terminus, and one less residue at the C-terminus than vertebrate cytochromes c. The region of residues 70-80 is strictly conserved, as is histidine at position 18. Position 26 is occupied by an asparagine residue, in contrast to histidine which occurs at this location in most of the known sequences of mammalian-type cytochromes c. 4. In contrast to some other fungal and plant cytochromes c of known primary structures, the Ustilago cytochrome c molecule does not contain trimethyl-lysine. 5. The sequence of Ustilago cytochrome c differs from the sequences of human, horse, chicken, tuna, wheat, and baker's yeast proteins at loci 47, 43, 44, 44 and 38 respectively.     

Amino acid sequences of the two heme c-binding sites of Pseudomonas cytochrome-c peroxidase

The amino acid sequences of the two heme c-containing tryptic peptides of Pseudomonas cytochrome-c peroxidase have been determined. The tryptic peptides were isolated from two cyanogen bromide fragments of the protein. Both heme-binding sites have the Cys-X-Y-Cys-His structure characteristic of c-type cytochromes. The sequences of the two peptides show distinct homology with each other, suggesting the occurrence of gene doubling during evolution of the protein molecule. The function of the heme c moieties in the catalytic cycle of the enzyme is discussed on the basis of their homology with the proximal histidine region of peroxidase (horseradish peroxidase and yeast cytochrome-c peroxidase) and cytochromes (horse cytochrome c and Pseudomonas cytochrome c-551).     

Leishmania major peroxidase is a cytochrome c peroxidase

Leishmania major peroxidase (LmP) exhibits both ascorbate and cytochrome c peroxidase activities. Our previous results illustrated that LmP has a much higher activity against horse heart cytochrome c than ascorbate, suggesting that cytochrome c may be the biologically important substrate. To elucidate the biological function of LmP, we have recombinantly expressed, purified, and determined the 2.08 ? crystal structure of L. major cytochrome c (LmCytc). Like other types of cytochrome c, LmCytc has an electropositive surface surrounding the exposed heme edge that serves as the site of docking with redox partners. Kinetic assays performed with LmCytc and LmP show that LmCytc is a much better substrate for LmP than horse heart cytochrome c. Furthermore, unlike the well-studied yeast system, the reaction follows classic Michaelis-Menten kinetics and is sensitive to an increasing ionic strength. Using the yeast cocrystal as a control, protein-protein docking was performed using Rosetta to develop a model for the binding of LmP and LmCytc. These results suggest that the biological function of LmP is to act as a cytochrome c peroxidase.     

Definition of the interaction domain for cytochrome c on cytochrome c oxidase. III. Prediction of the docked complex by a complete, systematic search

The electron transfer complex between bovine cytochrome c oxidase and horse cytochrome c has been predicted with the docking program DOT, which performs a complete, systematic search over all six rotational and translational degrees of freedom. Energies for over 36 billion configurations were calculated, providing a free-energy landscape showing guidance of positively charged cytochrome c to the negative region on the cytochrome c oxidase surface formed by subunit II. In a representative configuration, the solvent-exposed cytochrome c heme edge is within 4 A of the indole ring of subunit II residue Trp(104), indicating a likely electron transfer path. These two groups are surrounded by a small, hydrophobic contact region, which is surrounded by electrostatically complementary hydrophilic interactions. Cytochrome c/cytochrome c oxidase interactions of Lys(13) with Asp(119) and Lys(72) with Gln(103) and Asp(158) are the most critical polar interactions due to their proximity to the hydrophobic region and exclusion from bulk solvent. The predicted complex matches previous mutagenesis, binding, and time-resolved kinetics studies that implicate Trp(104) in electron transfer and show the importance of specific charged residues to protein affinity. Electrostatic forces not only enhance long range protein/protein association; they also predominate in short range alignment, creating the transient interaction needed for rapid turnover.     

Compressing the free energy range of substructure stabilities in iso‐1‐cytochrome c

Evolutionary conservation of substructure architecture between yeast iso-1-cytochrome c and the well-characterized horse cytochrome c is studied with limited proteolysis, the alkaline conformational transition and global unfolding with guanidine-HCl. Mass spectral analysis of limited proteolysis cleavage products for iso-1-cytochrome c show that its least stable substructure is the same as horse cytochrome c. The limited proteolysis data yield a free energy of 3.8 ± 0.4 kcal mol⁻¹ to unfold the least stable substructure compared with 5.05 ± 0.30 kcal mol⁻¹ for global unfolding of iso-1-cytochrome c. Thus, substructure stabilities of iso-1-cytochrome c span only ∼1.2 kcal mol⁻¹ compared with ∼8 kcal mol⁻¹ for horse cytochrome c. Consistent with the less cooperative folding thus expected for the horse protein, the guanidine-HCl m-values are ∼3 kcal mol⁻¹M⁻¹ versus ∼4.5 kcal mol⁻¹M⁻¹ for horse versus yeast cytochrome c. The tight free energy spacing of the yeast cytochrome c substructures suggests that its folding has more branch points than for horse cytochrome c. Studies on a variant of iso-1-cytochrome c with an H26N mutation indicate that the least and most stable substructures unfold sequentially and the two least stable substructures unfold independently as for horse cytochrome c. Thus, important aspects of the substructure architecture of horse cytochrome c, albeit compressed energetically, are preserved evolutionally in yeast iso-1-cytochrome c.