Substitutions engineered by chemical synthesis at three conserved sites in mitochondrial cytochrome c. Thermodynamic and functional consequences

Analogues of the 39-residue CNBr fragment of horse cytochrome c (66-104) have been prepared by total chemical synthesis. Conformationally assisted ligation of these peptides with the native cytochrome c fragment 1-65 (homoserine lactone form) occurred in high yield. Semisynthetic protein molecules of the expected molecular weight were obtained that had folded structures similar to the native molecule as shown by spectral properties and by cross-reactivity with a panel of monoclonal antibodies sensitive to the three-dimensional integrity of cytochrome c. Point mutations were introduced into the horse sequence at three strongly conserved sites: Tyr67, Thr78, and Ala83. The contributions of these 3 residues to the stability of the heme crevice were estimated by titration of the 695 nm absorption due to coordination of ferric iron by the sixth ligand methionine sulfur. The roles of these residues in catalysis of electron transfer and in establishing the value of the redox potential of cytochrome c were also investigated. The hydroxyl group of Tyr67 modulates the spectral properties of the heme and has a profound influence on its redox properties, but hydrogen bonding involving this phenolic hydroxyl does not stabilize the heme crevice. In contrast, we find that Thr78 is strongly stabilizing and that asparagine is not an adequate substitute for this residue because of the greater entropic cost of burying its side chain. The low biological activity of analogues modified at this position, despite normal redox potentials, imply a role for Thr78 in the electron transfer mechanism. The replacement of Ala83 by proline induces a similar phenomenon. An involvement of this residue in the catalysis of electron transfer provides an explanation of the low reactivity of plant mitochondrial cytochromes c in mammalian redox systems.     

The oxidation-state-dependent ATP-binding site of cytochrome c. Implication of an essential arginine residue and the effect of occupancy on the oxidation-reduction potential.

Arg-91 is not part of the active site of cytochrome c that mediates binding and electron transfer, yet it is absolutely conserved in eukaryotic cytochromes c, indicating a special function. The physicochemical properties of analogues are unaffected by the modification of this residue, so they can be used with confidence to study the role of Arg-91. We have established limiting conditions under which this residue alone is specifically modified by cyclohexane-1,2-dione, and have subsequently shown that ATP, and to a lesser extent ADP or Pi, protects it from the action of the reagent in an oxidation-state-dependent manner. These observations strongly support the idea that this site exerts a controlling influence on cytochrome c activity in the electron transport or other cellular redox systems, and we have commenced a study of how that influence might operate. We find that the redox potentials of both cytochrome c and analogue are little affected by changing ATP or Pi concentrations.     

The semisynthesis of fragments corresponding to residues 66-104 of horse heart cytochrome c.

We describe the N epsilon-acetimidylation of horse heart cytochrome c with retention of biological activity, the cleavage of the modified protein by CNBr, the separation of the fragments, and their further side-chain protection. We describe the manipulation of the amino acid sequences of the fragments by stepwise semisynthetic methods. We have prepared fragments corresponding to residues 66-78 and 66-79 of the protein, as well as the [Asp66] analogue of fragment 66-79. We have prepared the natural sequence and the [o-fluoro-Phe82] analogue of the fragment corresponding to residues 81-104 of the protein, and the [N epsilon-trifluoroacetyl-Lys79], the [N epsilon-dinitrophenyl-Lys79] and the [S-acetamidomethyl-Cys79] analogues of fragment 79-104, and the [N epsilon-Cbz-Lys81] analogue of fragment 80-104. We have coupled back the fragments of natural sequence to form a semisynthetic fragment corresponding to residues 66-104 of the protein. Modified fragments were also coupled to give analogues of the 66-104-residue sequence. In every case the homoserine residue representing methionine-80 was removed from the C-terminus of the 66-80-residue fragment and replaced by methionine on the N-terminus of the 81-104 residue fragment during the preparation of the fragments for coupling. The semisynthetic fragments are ready for specific deprotection and further coupling. We have coupled one such fragment to the (1-65)-peptide to produce semisynthetic [Hse65]cytochrome c. The product has satisfactory characteristics on chemical analysis, and on assay of its biological activity.     

Formation of a biologically active, ordered complex from two overlapping fragments of cytochrome c.

A noncovalent complex of the apoprotein (1-104) and cyanogen bromide heme fragment containing residues 1 to 65, (1-65) H, has been prepared from horse heart cytochrome c. Conditions under which the redundant portions of the ferrous complex can be removed by limited trypsin digestion have been devised. The complementing fragments have been isolated from the derived complexes and four apofragments and one heme fragment have been identified in the amino acid sequence of cytochrome c. They are (39-104), (40-104), (54-104), (56-104), and (1-53)H. The formation of an ordered ferric complex composed of one heme fragment and one apofragment for the cases (1-53)H (39-104), (1-53)H-(40-104), (1-53)H-(54-104), and (1-53)H-(56-104) has been demonstrated by the quenching of the tryptophan 59 fluorescence and the regain of biological activity in a cytochrome b2 assay. The apparent dissociation constant has been estimated as less than 3 X 10(-7) M in all the aforementioned cases. Thus, the region (between residues 38 and 57) of the amino acid sequence permissible for cleavage without disruption of the ordered structure indicated by the present in vitro experiments corresponds to that (between residues 38 and 57) evolutionally deleted in the three-dimensional structure of Pseudomonas aeruginosa cytochrome c551 discovered by Dickerson et al. (Dickerson, R.E., Timkovich, R., and Almassy, R.J. (1976) J. Mol. Biol. 100, 473-491).     

The effect of complete or specific partial acetimidylation on the biological properties of cytochrome c and cytochrome c-T.

The biological consequences of acetimidylation of all 19 epsilon-amino groups of horse cytochrome c are a slight decrease in both the redox potential of the protein and its ability to stimulate oxygen uptake in the cytochrome c-depleted-mitochondria assay. Examination of a number of specific partially acetimidylated analogues and acetimidylated cytochromes c of other species has shown that the changes in biological properties, which are associated with a slight structural change as monitored by n.m.r. spectroscopy [Boswell, Moore, Williams, Harris, Wallace, Bocieck & Welti (1983) Biochem. J. 213, 679-686], appear to stem from modification of residues in a restricted region of the sequence. The failure of the redox potential of Saccharomyces cerevisae cytochrome c to be affected by acetimidylation suggests that it is lysine-53, absent from that species, that is the sensitive residue.     

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.     

Semisynthetic analogs of cytochrome c reconstructed from natural and synthetic peptides.

A biologically active semisynthetic hybrid of horse heart cytochrome c has been prepared by combining the heme peptide 1 through 65 (HP 1-65), prepared by CNBr cleavage of natural cytochrome c, with a semisynthetic peptide corresponding to positions 66 through 104. A fully protected synthetic peptide 66--79 was prepared by a modified solid phase peptide synthesis procedure and was converted to its N-hydroxysuccinimide ester. A peptide corresponding to residues 81--104 of cytochrome c was also isolated from the CNBr cleavage mixture and its epsilon-amino groups and tyrosyl hydroxyl group were protected selectively with the t-butyloxycarbonyl group. This partially protected peptide was reacted with t-butyloxycarbonyl methionine N-hydroxysuccinimide ester to give a derivative having methionine at position 80. This product was deprotected, purified and then t-butyloxycarbonyl groups were again introduced specifically on the epsilon-amino groups to give the peptide, Boc(Lys,Tyr)80--104. A semisynthetic peptide corresponding to residues 66 through 104 of cytochrome c was prepared by condensing the synthetic peptide 66--79 N-hydroxysuccinimide ester with t-butyloxycarbonyl (Lys,Tyr)80--104. The semisynthetic product was deprotected, purified and combined under anaerobic conditions with a heme peptide, HP 1-65, that was isolated from the products of CNBr cleavage of native cytochrome c. The reconstituted semisynthetic cytochrome c was purified by ion exchange chromatography and was shown to have the same oxygen uptake as native cytochrome c when assayed in the succinate oxidase system.     

Covalent structure of the membranous segment of horse cytochrome b5. Chemical cleavage of the native hemoprotein

The complete covalent structure of the membranous segment of horse liver cytochrome b5 has been determined. This peptide spans residues 91 to 133 of the cytochrome molecule, and contains the segment responsible for the association of the hemoprotein with microsomal or synthetic vesicles. Two peptides, residues 91 to 127 and 128 to 133, comprising the entire membranous moiety were isolated from a tryptic digest of urea-denatured apoprotein. The membranous segment (residues 91 to 127) could be separated from all other tryptic peptides by a single gel filtration step. Trypsin digestion of succinylated cytochrome produced similar peptides, residues 89 to 127 and 128 to 133. The covalent structures for residues 89 to 127 and 128 to 133 were derived from automated sequenator analysis of tryptic peptides. Chemical cleavage at tryptophanyl, or methionyl residues, or both, by the method of Ozols and Gerard ((1977) J. Biol. Chem. 252, 5986-5989) provided the overlapping peptides from which the following unique sequence was deduced: (formula: see text).     

Alkylamine derivatives of cytochrome c. Comparison with other lysine-modified analogues illuminates structure/function relations in the protein

For investigations of the functional roles of the lysine residues of cytochrome c, analogues in which these residues are modified without charge loss are highly desirable. The 19 lysine residues of the horse heart protein have been modified by reductive alkylation. Two analogues were prepared, using formaldehyde and acetone as the dialkylating and monoalkylating reagent respectively. Modification was shown to be clean and quantitative. Characterisation of the alkylamine derivatives by physicochemical measurements and biological activity determinations was carried out. The potential of these analogues in structure/function studies of cytochrome c is discussed. It is illustrated by their use, in conjunction with other lysine-modified derivatives, to investigate the extent to which surface charge determines redox potential, and to study the physicochemical changes that accompany rising pH. In the latter case the observed phenomena are not as closely correlated as previously thought, suggesting that there is a complex set of rearrangements of structure underlying the functional changes. The data confirm that modification of the lysine residues influences these changes. These residues participate in numerous surface intramolecular links, so the lack of correlation may be explained if each of the changing parameters were under the influence of a different set of residues. However, neither a lysine residue, nor a histidine residue directly displaces methionine from the sixth coordination position of the haem iron at alkaline pH.     

A tale of two charges: distinct roles for an acidic and a basic amino acid in the structure and function of cytochrome c.

Cytochrome c is a small electron transport protein found in the intermembrane space of mitochondria. As it interacts with a number of different physiological partners in a specific fashion, its structure varies little over eukaryotic evolutionary history. Two highly conserved residues found within its sequence are those at positions 13 and 90 (numbering is based on the standard horse cytochrome c); with single exceptions, residue 13 is either Lys or Arg, and residue 90 is either Glu or Asp. There have been conflicting views on the roles to be ascribed to these residues, particularly residue 13, so the functional properties of a number of site-directed mutants of Saccaromyces cerevisiae iso-1 cytochrome c have been examined. Results indicate that the two residues do not interact specifically with each other; however, residue 13 (Arg) is likely to be involved in interactions between cytochrome c and other electrostatically oriented physiological partners (intermolecular), whereas residue 90 (Asp) is involved in maintaining the intrinsic structure and stability of cytochrome c (intramolecular). This is supported by molecular dynamics simulations carried out for these mutants where removal of the negative charge at position 90 leads to significant shifts in the conformations of neighboring residues, particularly lysine 86. Both charged residues appear to exert their effects through electrostatics; however, biological activity is significantly more sensitive to substitutions of residue 13 than of residue 90.     

L-serine binds to arginine-148 of the beta 2 subunit of Escherichia coli tryptophan synthase

Inactivation of the beta 2 subunit and of the alpha 2 beta 2 complex of tryptophan synthase of Escherichia coli by the arginine-specific dicarbonyl reagent phenylglyoxal results from modification of one arginyl residue per beta monomer. The substrate L-serine protects the holo beta 2 subunit and the holo alpha 2 beta 2 complex from both inactivation and arginine modification but has no effect on the inactivation or modification of the apo forms of the enzyme. This result and the finding that phenylglyoxal competes with L-serine in reactions catalyzed by both the holo beta 2 subunit and the holo alpha 2 beta 2 complex indicate that L-serine and phenylglyoxal both bind to the same essential arginyl residue in the holo beta 2 subunit. The apo beta 2 subunit is protected from phenylglyoxal inactivation much more effectively by phosphopyridoxyl-L-serine than by either pyridoxal phosphate or pyridoxine phosphate, both of which lack the L-serine moiety. The phenylglyoxal-modified apo beta 2 subunit binds pyridoxal phosphate and the alpha subunit but cannot bind L-serine or L-tryptophan. We conclude that the alpha-carboxyl group of L-serine and not the phosphate of pyridoxal phosphate binds to the essential arginyl residue in the beta 2 subunit. The specific arginyl residue in the beta 2 subunit which is protected by L-serine from modification by phenyl[2-14C]glyoxal has been identified as arginine-148 by isolating a labeled cyanogen bromide fragment (residues 135-149) and by digesting this fragment with pepsin to yield the labeled dipeptide arginine-methionine (residues 148-149). The primary sequence near arginine-148 contains three other basic residues (lysine-137, arginine-141, and arginine-150) which may facilitate anion binding and increase the reactivity of arginine-148. The conservation of the arginine residues 141, 148, and 150 in the sequences of tryptophan synthase from E. coli, Salmonella typhimurium, and yeast supports a functional role for these three residues in anion binding. The location and role of the active-site arginyl residues in the beta 2 subunit and in two other enzymes which contain pyridoxal phosphate, aspartate aminotransferase and glycogen phosphorylase, are compared.     

Definition of cytochrome c binding domains by chemical modification. II. Identification and properties of singly substituted carboxydinitrophenyl cytochromes c at lysines 8, 13, 22, 27, 39, 60, 72, 87, and 99.

Sensitive thin layer peptide mapping is employed to establish the identity and the homogeneity of eight singly substituted 4-carboxy-2,6-dinitrophenyl derivatives of horse cytochrome c. Seven of the components, all of greater than 95% homogeneity, are modified at lysyl residues 13, 72, 87, 8, 27, 39, and 60. The eighth component is a mixture of derivatives at lysines 22 and 99. The positions of the modified residues were confirmed by the amino acid analysis and Edman sequential degradation of the CDNP-peptides. Physiochemical properties characteristic of cytochrome c are unchanged in the chemically modified products examined. These properties, that include the proton NMR spectrum, are sensitive probes of the polypeptide organization surrounding the heme prosthetic group. The lack of any discernable changes indicates that modification of the epsilon-amino groups on the surface of cytochrome c does not perturb the overall structure of the protein. The widespread distribution of the modifications on the surface of the molecule, together with the homogeneity and native conformation of the CDNP-derivatives make them well suited for assessing the effects of changes in the charge topography on the electron transfer activity of cytochrome c.     

Membrane location of spin-labeled cytochrome c determined by paramagnetic relaxation agents

The mitochondrial protein horse heart cytochrome c was specifically spin-labeled with succinimidyl-2,2,5, 5-tetramethyl-3-pyrroline-1-oxyl-carboxylate on different lysine residues at positions 86, 87, 72, 8, or 25, respectively. Site-specifically labeled species were separated chromatographically and identified by peptide sequencing of tryptic digests. The monolabeled protein was bound to negatively charged phospholipid membranes composed of dioleoylphosphatidylglycerol, and the accessibility of the spin-labeled lysine residues to lipid-soluble molecular oxygen and to lipid-impermeant chromium maltolate was determined from the saturation properties of the ESR spectra. The accessibilities of the spin-labeled proteins relative to those obtained for phospholipids spin-labeled in the headgroup region, in the presence of unlabeled protein, identify the position of the spin-labeled lysine residues relative to the phospholipid bilayer surface. We have found that cytochrome c does not penetrate into the membrane interior and that the active side of cytochrome c in the protein-membrane interaction is the side on which lys86, lys87, and lys72 are located.     

The conformation of eukaryotic cytochrome c around residues 39, 57, 59 and 74.

1H-n.m.r. studies of horse, tuna, Candida krusei and Saccharomyces cerevisiae cytochromes c showed that each of the proteins contains a similar cluster of residues at the bottom of the protein that assists in shielding the haem from the solvent. The relative positions of the residues forming these clusters vary continuously with temperature, and they change with the change in protein redox state. This conformational heterogeneity is discussed with reference to the conformational flexibility of cytochrome c around residues 57, 59 and 74. Spectroscopic measurements of pKa values for Lys-55 (horse and tuna cytochromes c) and His-33 and His-39 (C. krusei and S. cerevisiae cytochromes c) are in excellent agreement with expectations based on chemical-modification studies of horse cytochrome c. [Bosshard & Zürrer (1980) J. Biol. Chem. 255, 6694-6699] and on the X-ray-crystallographic structure of tuna cytochrome c [Takano & Dickerson (1981) J. Mol. Biol. 153, 79-94, 95-115].     

ATP binding to cytochrome c diminishes electron flow in the mitochondrial respiratory pathway.

Eukaryotic cytochrome c possesses an ATP-binding site of substantial specificity and high affinity that is conserved between highly divergent species and which includes the invariant residue arginine91. Such evolutionary conservatism strongly suggests a physiological role for ATP binding that demands further investigation. We report the preparation of adducts of the protein and the affinity labels 8-azido adenosine 5'-triphosphate, adenosine 5'-triphosphate-2',3'-dialdehyde, and 5'-p-fluorosulfonylbenzoyladenosine. The two former reagents were seen to react at the arginine91-containing site, yet the reaction of the latter, although specific, occurred elsewhere, suggesting caution is necessary in its use. None of the adducts displayed significant modification of global structure, stability, or physicochemical properties, leading us to believe that the 8-N3-ATP and oATP adducts are good stabilized models of the noncovalent interaction; yet modification led to significant, and sometimes pronounced, effects on biological activity. We therefore propose that the role of ATP binding to this site, which we have shown to occur when the phosphorylation potential of the system is high under the equivalent of physiological conditions, is to cause a decrease in electron flow through the mitochondrial electron transport chain. Differences in the degree of inhibition produced by differences in adduct chemistry suggest that this putative regulatory role is mediated primarily by electrostatic effects.     

A relationship between prokaryote and eukaryote observed in Nitrobacter agilis cytochromes AA3 and C

Cytochrome aa3 (cytochrome c oxidase) and cytochrome c were purified from Nitrobacter agilis, and some of their properties were compared with those of the respective counterparts of eukaryote from the evolutionary point of view. N. agilis cytochrome aa3 has many functional and structural properties similar to those of eukaryotic cytochrome aa3, although its molecule is composed of only two kinds of subunits unlike the eukaryotic cytochrome which is composed of 7 kinds of subunits. N. agilis cytochrome c is homologous to eukaryotic cytochrome c; 50 amino acid residues of the bacterial cytochrome c are identical with those of horse cytochrome c. It reacts with yeast cytochrome c peroxidase as rapidly as eukaryotic cytochrome c does. So far as based on the molecular features of cytochromes aa3 and c, N. agilis appears to be one of the organisms which may link in evolution prokaryote to eukaryote.     

Structural role of the tyrosine residues of cytochrome c.

The tertiary structures of horse, tuna, Neurospora crassa, horse [Hse65,Leu67]- and horse [Hse65,Leu74]-cytochromes c were studied with high-resolution 1H n.m.r. spectroscopy. The amino acid sequences of these proteins differ at position 46, which is occupied by phenylalanine in the horse proteins but by tyrosine in the remaining two, and at positions 67, 74 and 97, which are all occupied by tyrosine residues in horse and tuna cytochrome c but in the other proteins are substituted by phenylalanine or leucine, though there is only one such substitution per protein. The various aromatic-amino-acid substitutions do not seriously affect the protein structure.     

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.     

Comparing substrate specificity between cytochrome c maturation and cytochrome c heme lyase systems for cytochrome c biogenesis

Hemes c are characterized by their covalent attachment to a polypeptide via a widely conserved CXXCH motif. There are multiple biological systems that facilitate heme c biogenesis. System I, the cytochrome c maturation (CCM) system, is found in many bacteria and is commonly employed in the maturation of bacterial cytochromes c in Escherichia coli-based expression systems. System III, cytochrome c heme lyase (CCHL), is an enzyme found in the mitochondria of many eukaryotes and is used for heterologous expression of mitochondrial holocytochromes c. To test CCM specificity, a series of Hydrogenobacter thermophilus cytochrome c(552) variants was successfully expressed and matured by the CCM system with CX(n)CH motifs where n = 1-4, further extending the known substrate flexibility of the CCM system by successful maturation of a bacterial cytochrome c with a novel CXCH motif. Horse cytochrome c variants with both expanded and contracted attachment motifs (n = 1-3) were also tested for expression and maturation by both CCM and CCHL, allowing direct comparison of CCM and CCHL substrate specificities. Successful maturation of horse cytochrome c by CCHL with an extended CXXXCH motif was observed, demonstrating that CCHL shares the ability of CCM to mature hemes c with extended heme attachment motifs. In contrast, two single amino acid mutants were found in horse cytochrome c that severely limit maturation by CCHL, yet were efficiently matured with CCM. These results identify potentially important residues for the substrate recognition of CCHL.     

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.