Determinants of cytochrome c pro-apoptotic activity. The role of lysine 72 trimethylation.

Cytochrome c released from vertebrate mitochondria engages apoptosis by triggering caspase activation. We previously reported that, whereas cytochromes c from higher eukaryotes can activate caspases in Xenopus egg and mammalian cytosols, iso-1 and iso-2 cytochromes c from the yeast Saccharomyces cerevisiae cannot. Here we examine whether the inactivity of the yeast isoforms is related to a post-translational modification of lysine 72, N-epsilon-trimethylation. This modification was found to abrogate pro-apoptotic activity of metazoan cytochrome c expressed in yeast. However, iso-1 cytochrome c lacking the trimethylation modification also was devoid of pro-apoptotic activity. Thus, both lysine 72 trimethylation and other features of the iso-1 sequence preclude pro-apoptotic activity. Competition studies suggest that the lack of pro-apoptotic activity was associated with a low affinity for Apaf-1. As cytochromes c that lack apoptotic function still support respiration, different mechanisms appear to be involved in the two activities.

The source of heterogeneity in the heme vicinity of ferricytochrome c.

Heterogeneity in the heme vicinity of ferricytochrome c was reported to be detectable by a split of the NMR signal of the heme methyl 3 group [P.D. Burns and G.N. La Mar, J. Am. Chem. Soc. 101 (1979) 5844]. Using cytochrome c mutants and computer simulations of the native and mutated cytochromes, the source of this heterogeneity is found to originate from the His-33 residue motions. The H33F mutation abolished the NMR split and computer simulations of the H33F mutant revealed a narrower distribution of fluctuations of the radius of gyration, suggesting a more rigid structure due to the mutation. The stabilization of the mutant was further demonstrated by a reduction in the H33F mutant of 4 Kcal/mol in the calculated interaction energy between residue 33 and the rest of the cytochrome, in keeping with known experimental results.

Using entropies of reaction to predict changes in protein stability: tyrosine-67-phenylalanine variants of rat cytochrome c and yeast Iso-1 cytochromes c.

Using the voltammetric method of square-wave voltammetry, a direct electrochemical examination was made of the wild type and Tyr67Phe mutant of both rat cytochrome c and yeast iso-1-cytochrome c. In addition to determining the equilibrium reduction potential (E0') for each cytochrome, the entropy of reaction, deltaS0'(Rxn)(deltaS0'(Rxn) = S0'(Red) - S0'(Ox)), for the reduction process was determined via the non-isothermal method. Having determined deltaS0'(Rxn) and E0', deltaH0' was calculated. For rat cytochrome c, it was found that deltaS0'(Rxn) = -43 J mol(-1) K(-1) for the wild type and -53 J mol(-1) K(-1) for the Tyr67Phe variant, with the deltaH0' for both the wild type and variant nearly identical, indicating that the changes in reduction potential and probably stability are due to changes in deltaS0'(Rxn). In contrast the measured deltaS0'(Rxn) for yeast iso-1-cytochrome c demonstrated significant changes in both entropic and enthalpic contributions in going from wild type to mutant cytochrome c. The entropy of reaction provides information regarding the relative degree of solvation, and very likely the degree of compactness, of the oxidized state versus the reduced state of the redox protein. A thermodynamic scheme and stability derivation are presented that show how the entropies of reaction of wild type versus variant cytochromes contribute to and predict changes in stability in going from oxidized to reduced protein. For yeast iso-1-cytochrome c, the thermodynamically predicted change in stability was very close to the experimentally observed value, based on previous differential scanning calorimetric stability measurements. While such data is not available for rat cytochrome c, consideration of the enormously increased local stability of the rat oxidized cytochrome c variant predicts that the reduced rat variant will be even more stable than the already stabilized oxidized variant.

Cytochrome c heme lyase activity of yeast mitochondria.

A highly efficient in vitro system was established for measuring by high performance liquid chromatography the formation of holocytochrome c by yeast mitochondria. Holocytochrome c formation required reducing agents, of which dithiothreitol was the most effective. With biosynthetically made, pure Drosophila melanogaster apocytochrome c and Saccharomyces cerevisiae mitochondria, the activity of cytochrome c heme lyase amounted to about 800 fmol min-1 mg-1 mitochondrial protein. The kinetics were typical Michaelis-Menten (Km approximately 1 nM), as were those of mitoplasts with broken outer membranes (Km approximately 3 nM). As tested with mitoplasts, holocytochromes c from a range of species were found to be competitive inhibitors of heme lyase at physiological concentrations, providing a mechanism for controlling this concentration in vivo. Apocytochrome c associated with yeast mitochondria in two phases of Kd approximately 2 x 10(-10) and 10(-8) M, respectively, whereas mitoplasts had lost the high affinity binding. A site-directed mutant of apocytochrome c (lysines 5, 7, and 8 replaced by glutamine, glutamic acid, and asparagine) was found to be converted to holocytochrome c (Km approximately 3.3 nM; maximal activity unchanged), even though the mutations completely eliminated the high affinity binding. Thus, the high affinity binding of apocytochrome c to mitochondria is not directly related to holocytochrome c formation.

Direct voltammetric observation of redox driven changes in axial coordination and intramolecular rearrangement of the phenylalanine-82-histidine variant of yeast iso-1-cytochrome c.

Direct square-wave and cyclic voltammetric electrochemical examination of the yeast iso-1-cytochrome c Phe82His/Cys102Ser variant revealed the intricacies of redox driven changes in axial coordination, concomitant with intramolecular rearrangement. Electrochemical methods are ideally suited for such a redox study, since they provide a direct and quantitative visualization of specific dynamic events. For the iso-1-cytochrome c Phe82His/Cys102Ser variant, square-wave voltammetry showed that the primary species in the reduced state is the Met80-Fe2+-His18 coordination form, while in the oxidized state the His82-Fe3+-His18 form predominates. The addition or removal of an electron to the appropriate form of this variant serves as a switch to a new molecular form of the cytochrome. Using the 2 x 2 electrochemical mechanism, simulations were done for the cyclic voltammetry experiments at different scan rates. These, in turn, provided relative rate constants for the intramolecular rearrangement/ligand exchange and the equilibrium redox potentials of the participating coordination forms: kb,AC = 17 s-1 for Met80-Fe3+-His18 --> His82-Fe3+-His18 and kf,BD > 10 s-1 for His82-Fe2+-His18 --> Met80-Fe2+-His18; E0' = 247 mV for Met80-Fe3+/2+-His18 couple, E0' = 47 mV for His82-Fe3+/2+-His18 couple, and E0' = 176 mV for the cross-reaction couple, His82-Fe3+-His18 + e- --> Met80-Fe2+-His18. Thermodynamic parameters, including the entropy of reaction, DeltaS0'Rxn, were determined for the net reduction/rearrangement reaction, His82-Fe3+-His18 + e- --> Met80-Fe2+-His18, and compared to those for wild-type cytochrome, Met80-Fe3+-His18 + e- --> Met80-Fe2+-His18. For the Phe82His variant mixed redox couple, DeltaS0'Rxn = -80 J/mol.K compared to DeltaS0'Rxn = -52 J/mol.K for the wild-type cyt c couple without rearrangement. Comparison of these entropies indicates that the oxidized His82-Fe3+-His18 form is highly disordered. It is proposed that this high level of disorder facilitates rapid rearrangement to Met80-Fe2+-His18 upon reduction.

The nature of the thermal equilibrium affecting the iron coordination of ferric cytochrome c.

In cytochrome c, ligation of the heme iron by the methionine-80 sulfur plays a major role in determining the structure and the thermodynamic stability of the protein. In the ferric state, this bond is reversibly broken by moderately acid or alkaline pH's (pK's 2.5 and 9.4, respectively) and by exogenous ligands. NMR studies of horse ferricytochrome c in which the Met-65 and Met-80 methyl groups were chemically enriched with 13C demonstrate that, at 59 degrees C, a temperature at which the protein is still folded, the sulfur-iron bond is already partially broken. This structural change corresponds to the reversible disappearance upon moderate heating of the 695 nm band, characteristic of the sulfur-iron coordination of this protein. The thermal effect results from a shift in the alkaline pK from 9.4 at 25 degrees C to 8.2 at 59 degrees C. The exchange rate from iron-bound to free methionine-80 at 59 degrees C is 1.8 s-1, as measured by saturation transfer experiments. The free and bound methionine-80 epsilon-methyl groups in the 1H spectrum are assigned as (1.87, 2.25) and -21.43, respectively; in the 13C spectrum they are assigned as 15.6 and 12.8, respectively (all these values are in ppm from 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium salt).

A chemical modification of cytochrome-c lysines leading to changes in heme iron ligation.

Although 13 lysines of horse cytochrome c are invariant, and three more are extremely conserved, the modification of their side-chain epsilon-amino groups by beta-thiopropionylation caused important changes in protein properties for only three of them; lysines 72,73 and 79. Optical spectroscopy, electron and nuclear paramagnetic resonance, electron spin echo envelope modulation, and molecular weight studies, as well as the unique features of their reaction with cytochrome-c oxidase, indicate that in the oxidized state the modification of these lysines resulted in equilibria between two different states of iron ligation: the native state, in which the metal is coordinated by the methionine-80 sulfur, and a new state in which this ligand is displaced by the sulfhydryl groups of the elongated side chains. The reduction potentials of the TP Lys-72 and the TP Lys-79 derivatives were 201 and 196 millivolt, respectively, indicating that the equilibria favored the sulfhydryl ligated state by 1.5 and 1.7 kcal/mol, respectively. In the ferric state, the protein modified at lysine 72 remained stable as a monomer, but that modified at lysine 73 dimerized rapidly through disulfide bond formation, while the TP Lys-79 cytochrome c dimerized with a half-time of approx. 3 h, both recovering the native-like iron ligation. By contrast, in the ferrous state the monomeric state and the native ligation were preserved in all cases, indicating that the affinity of the cytochrome-c ferrous iron for the methionine-80 sulfur is particularly strong. The dimerized derivatives lost most, but not all, of the capability of the native protein for electron transfer from ascorbate-TMPD to cytochrome-c oxidase.

Beta-thiopropionyl cytochromes c modified at lysyl residues: preparation and characterization of the monosubstituted horse cytochromes c.

beta-Thiopropionyl derivatives of horse cytochrome c singly modified at each of 18 different lysine epsilon-amino groups have been prepared using sulfosuccinimidyl-2-(biotinamido)ethyl-1,3-dithiopropionate and purified to homogeneity by high-pressure liquid chromatography. These derivatives were characterized by determination of: (i) the location of the modification; (ii) reduction potentials; (iii) visible and NMR spectra: and by (iv) measurement of electron transfer activity with cytochrome-c oxidase. No significant changes in structure were indicated, except for the ferric forms of the derivatives modified at lysines 72, 73, and 79 which are discussed separately. The electron transfer activity of the beta-thiopropionyl cytochromes c with bovine heart cytochrome-c oxidase was decreased to extents dependent on the position of the modification. Aminoethylation, a secondary modification which reverses the charge change, restored the electron transfer rate to that observed with the unmodified cytochrome c, irrespective of the location of the primary modification. These results afford a direct experimental demonstration that alterations in kinetics with physiological electron transfer partners resulting from modifications which cause a change of the charge of surface side chains are solely due to the electrostatic effects. Of the many chemically modified cytochromes c prepared to date, the singly substituted beta-thiopropionyl cytochromes c are likely to be particularly useful as the thiol allows covalent linkage of any sulfhydryl-reactive reagent to a well-defined location on the protein surface by a simple procedure, even when the secondary modifier is relatively unstable, a crucial advantage not otherwise readily achieved.

The role of histidines 26 and 33 in the structural stabilization of cytochrome c.

Comparative studies of the importance of the two histidines of rat cytochrome c that are not ligands of the heme iron, for the stability of the protein, were carried out by site-directed mutagenesis. Histidine 26 was substituted by valine and the resulting effects on the stability of the Met-80-sulfur to heme iron bond to changes in pH and temperature, and of the global stability of the protein to unfolding in urea solutions, were measured. It is suggested that the loss of the hydrogen bond between the His-26 imidazole and the backbone amide of Asn-31 caused the observed decreases in local stability; and that, in addition, the elimination of the hydrogen bond between this imidazole and the carbonyl of Pro-44 resulted in an increase of the mobility of the lower loop (residues 41-47) on the right side of the protein and of its distance from the middle loop (residues 26-31), probably leading to greater hydration of the interior right side of the molecule. These changes resulted in a decrease in the global stability of the protein. Further mutation of Asn-52 to Ile led to a total recovery of the wild-type stability of the sulfur-iron bond, and a partial restoration of the global stability of the protein. Substitution of Phe for His-33 did not alter the sulfur-iron bond but caused a pronounced increase in the global stability of the protein. It is suggested that this effect results from hydrophobic interaction of the Phe-33 side chain with the lower loop on the right side of the protein. Such an interaction also explains the observation that the same mutation reversed the loss of global stability caused by substitution of Val to His-26, but did not restore the strength of the sulfur-iron bond that this mutation had brought about.

Major and minor epitopes on the self antigen mouse cytochrome c mapped by site-directed mutagenesis.

Variants of rat (mouse) cytochrome c, prepared by site-directed mutagenesis or represented by closely-related cytochromes c from different species, were employed to map the functional boundaries of a number of mouse monoclonal antibodies (mAb) specific for the major antigenic region on the self antigen (Ag) around residue 62 and the minor antigenic region around residue 44. The recombinant mouse cytochromes c tested were, unlike the tissue-derived Ag, trimethylated at position 72, and included the wild-type which was acetylated at the amino terminus, a variant that was unacetylated at the amino terminus, and variants with the following single amino acid residue replacements: V11I (valine to isoleucine at position 11), Q12M, A15S, A44P, F46Y, D50A, T58I and G89E. Of these, only the A44P variant affected the binding of mAb to the region previously localized to the vicinity of residue 44, thus confirming that assignment. Loss of the acetyl group at the amino terminus affected the binding of most of the mAb to the region around residue 62. The other mutations had little, if any, affect on mAb binding. The epitopes of mAb binding the region around residue 62 were shown in this study to have similar functional boundaries. This site on the self Ag, which encompasses at least three discontinuous segments of the polypeptide chain, is comparable in size to epitopes on other protein Ag that have been mapped by X-ray crystallography and is similar to an epitope in the corresponding region of the foreign Ag, horse cytochrome c, that has been mapped by hydrogen-deuterium exchange. In addition to the mAb binding the regions around residues 44 and 62, a third group of mouse cytochrome c-specific mAb known to be broadly reactive with mammalian cytochromes c and that represents a minor portion of the mAb was tested for binding the site-directed mutants of mouse cytochrome c. None of these mAb were affected by the mutations, indicating the presence of at least one more antigenic region on the self Ag in an area not encompassed by these mutations that is structurally highly conserved.

The low ionic strength crystal structure of horse cytochrome c at 2.1 A resolution and comparison with its high ionic strength counterpart.

BACKGROUND: Cytochrome c is an integral part of the mitochondrial respiratory chain. It is confined to the intermembrane space of mitochondria, and has the function of transferring electrons between its redox partners. Solution studies of cytochrome c indicate that the conformation of the molecule is sensitive to the ionic strength of the medium. RESULTS: The crystal structures of cytochromes c from several species have been solved at extremely high ionic strengths of near-saturated solutions of ammonium sulfate. Here we present the first crystal structure of ferricytochrome c at low ionic strength refined at 2.1 A resolution. In general, the structure has the same features as those determined earlier. However, there are some differences in both backbone and side-chain conformations in several areas. These areas coincide with those observed by NMR and resonance Raman spectroscopy to be sensitive to ionic strength. CONCLUSIONS: Neither ionic strength nor crystal-packing interactions have much influence on the conformation of horse cytochrome c. Nevertheless, some differences in the side-chain conformations at high and low ionic strengths may be important for understanding how the protein functions. Close examination of the gamma-turn (residues 27-29) conserved in cytochromes c leads us to propose the 'negative classical' gamma-turn to describe this unusual feature.

Enzymic activities of covalent 1:1 complexes of cytochrome c and cytochrome c peroxidase.

We have obtained several cysteine mutants in or around the cytochrome c peroxidase binding domain of rat and yeast iso-1 cytochrome c by site-directed mutagenesis. These cysteine residues were specifically labeled with the bifunctional photoactive cross-linker 4-azidophenacyl bromide (APB). 1:1 covalent complexes of cytochrome c peroxidase and cytochrome c were generated by cross-linking these specifically labeled cytochromes c to cytochrome c peroxidase, and the 1:1 complexes were purified. Steady-state kinetic studies of the purified 1:1 complexes with free yeast and horse cytochromes c showed the following: (1) Cytochrome c peroxidase has two distinct catalytic sites--a high-affinity and a low-affinity site. (2) Other than the difference in affinity, the binding of substrate at the low-affinity site is similar to that at the high-affinity site, with yeast cytochrome c interacting more strongly than the horse protein, the binding of both substrates being sensitive to ionic strength, and both sites able to transfer electrons. (3) HPLC chromatography of purified 1:1 complex showed multiple forms of 1:1 complexes, supporting the idea of multiple possible interactions between cytochrome c and the high-affinity site on cytochrome c peroxidase. (4) An allosteric or electrostatic effect exists between the two substrate binding sites, the binding of cytochrome c to the high-affinity site decreasing the binding affinity of the low-affinity site to cytochrome c. The higher the equilibrium binding affinity of the mutant cytochrome c to the peroxidase, the larger the apparent allosteric/electrostatic effect when that mutant protein is covalently bound to the high-affinity site of the enzyme. Furthermore, different locations of the covalently bound cytochrome c at the high-affinity site on the enzyme surface result in different degrees of allosteric/electrostatic effect. The presence of two active sites on the enzyme allows a simple interpretation of some of the differences in the steady-state kinetic behavior of cytochrome c peroxidase with horse and yeast iso-1 cytochrome c.

Circular dichroism studies of the binding of mammalian and non-mammalian cytochromes c to cytochrome c oxidase, cytochrome c peroxidase, and polyanions.

The effects of binding of Candida krusei, Drosophila melanogaster, horse, human, and rat cytochromes c to beef cytochrome c oxidase (ferrocytochrome c: oxygen oxidoreductase, EC 1.9.3.1) and yeast cytochrome c peroxidase (ferricytochrome c: hydrogen-peroxide oxidoreductase, EC 1.11.1.5) on their circular dichroism spectra were determined. The binding to cytochrome oxidase results in a positive increase in the ellipticities of the positive and negative Cotton effects at 404 nm and 417 nm of cytochrome c. The horse, human, and rat cytochromes c display less of an increase in the ellipticity of the positive Cotton effect at 404 nm, but more of a positive change in the negative Cotton effect at 417 nm than the C. krusei or D. melanogaster proteins. Interaction with yeast cytochrome c peroxidase elicits only a positive change in the ellipticity of the positive Cotton effect at 404 nm. No significant change is observed in the negative Cotton effect at 417 nm. Rat cytochrome c variants with a phenylalanine in place of tyrosine-67 and/or an alanine in place of proline-30 all display circular dichroism spectral changes upon binding to cytochrome c oxidase or cytochrome c peroxidase identical to those of the unaltered protein. The increase in ellipticity at 404 nm upon binding occurs even though replacement of tyrosine-67 results in the loss of the positive Cotton effect at this position. Polyglutamate and phosvitin complexes of cytochrome c show changes in the circular dichroism spectrum similar to those observed with cytochrome c peroxidase. However, the magnitudes of the spectral changes were considerably less. A model is proposed in which the main cause of the circular dichroism spectral changes observed upon complexation arise from the exclusion of solvent from the exposed front heme edge. According to this model, the exclusion of solvent changes the relative asymmetry of the environment of the electronic transitions of the heme prosthetic group of cytochrome c, resulting in observed circular dichroic effects.

Crystallization of wild-type and mutant ferricytochromes c at low ionic strength: seeding technique and X-ray diffraction analysis.

Ferricytochromes c were crystallized at low ionic strength by macroseeding techniques. Large crystals were grown by seed-induced self-nucleation which occurred anywhere in the drop, regardless of the location of the seed crystal. This unusual crystal-seeding method worked reproducibly in our hands, and X-ray quality crystals have been prepared of several ferricytochromes c: horse, rat (recombinant wild type), and two site-directed mutants of the latter, tyrosine 67 to phenylalanine (Y67F) and asparagine 52 to isoleucine (N52I). Crystals of any one of these four proteins could be used as seeds for the crystallization of any one of the others. All the crystals are of the same crystal form, with space group P2(1)2(1)2(1). There are two protein molecules per asymmetric unit. The crystals are stable in the X-ray beam and diffract to at least 2.0 A, resolution. Full crystallographic data sets have been collected from single crystals of all four proteins.

Effects of mutating Asn-52 to isoleucine on the haem-linked properties of cytochrome c.

Asn-52 of rat cytochrome c and baker's yeast iso-1-cytochrome c was changed to isoleucine by site-directed mutagenesis and the mutated proteins expressed in and purified from cultures of transformed yeast. This mutation affected the affinity of the haem iron for the Met-80 sulphur in the ferric state and the reduction potential of the molecule. The yeast protein, in which the sulphur-iron bond is distinctly weaker than in vertebrate cytochromes c, became very similar to the latter: the pKa of the alkaline ionization rose from 8.3 to 9.4 and that of the acidic ionization decreased from 3.4 to 2.8. The rates of binding and dissociation of cyanide became markedly lower, and the affinity was lowered by half an order of magnitude. In the ferrous state the dissociation of cyanide from the variant yeast cytochrome c was three times slower than in the wild-type. The same mutation had analogous but less pronounced effects on rat cytochrome c: it did not alter the alkaline ionization pKa nor its affinity for cyanide, but it lowered its acidic ionization pKa from 2.8 to 2.2. These results indicate that the mutation of Asn-52 to isoleucine increases the stability of the cytochrome c closed-haem crevice as observed earlier for the mutation of Tyr-67 to phenylalanine [Luntz, Schejter, Garber and Margoliash (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 3524-3528], because of either its effects on the hydrogen-bonding of an interior water molecule or a general increase in the hydrophobicity of the protein in the domain occupied by the mutated residues. The reduction potentials were affected in different ways; the Eo of rat cytochrome c rose by 14 mV whereas that of the yeast iso-1 cychrome c was 30 mV lower as a result of the change of Asn-52 to isoleucine.

The significance of denaturant titrations of protein stability: a comparison of rat and baker's yeast cytochrome c and their site-directed asparagine-52-to-isoleucine mutants.

The residue asparagine-52 of rat cytochrome c and baker's yeast iso-1-cytochrome c was mutated to isoleucine by site-directed mutagenesis, and the unfolding of the wild-type and mutant proteins in urea or guanidinium chloride solutions was studied. Whereas the yeast mutant cytochrome unfolded in 4-7 M urea with a rate constant (k) of 1.7 x 10(-2) s-1, the rat mutant protein unfolded with k = 5.0 x 10(-2) s-1, followed by a slow partial refolding with k = 5.0 x 10(-4) s-1. Denaturant titrations indicated that the mutation increased the stability of the yeast cytochrome by 6.3 kJ (1.5 kcal)/mol, while it decreased that of the rat protein by 11.7 kJ (2.8 kcal)/mol. These results probably reflect structural differences between yeast iso-1 and vertebrate cytochromes c in the vicinity of the Asn-52 side chain.

Crystallization of two monoclonal Fab fragments of similar amino-acid sequence bound to the same area of horse cytochrome c and interacting by potentially distinct mechanisms.

The mouse monoclonal antibodies (mAb), 2E5.G10 and 1F5.D1, are specific for horse cytochrome c and appear to bind the same epitope, since their heavy (H) and light (L) chains are functionally interchangeable. Comparison of the amino-acid sequences suggests that slightly different interactions may be involved in antigen recognition. In addition, the H chains differ at only a few amino-acid residues from the H chain of a rat cytochrome c-specific mAb suggesting that specificity for one protein over another may be determined by these amino-acid differences. To address these possibilities, the three-dimensional structures of the Fab portions of the mAb bound to cytochrome c are being determined by X-ray diffraction analysis. Here we describe the preparation and crystallization of the two complexes with horse cytochrome c. The complex of the Fab fragment of 2E5.G10 with horse cytochrome c yielded crystals of X-ray diffraction quality under two sets of conditions; in both the space group was P2(1). The corresponding complex of 1F5.D1 under one of these conditions crystallized in the P2(1)2(1)2(1) space group. Three-dimensional X-ray data for these two complexes have been collected with nominal resolutions of 2.86 and 2.48 A, respectively.

Immunoelectron microscopic localization of testicular and somatic cytochromes c in the seminiferous epithelium of the rat.

Somatic and testis-specific cytochromes c were localized ultrastructurally in the seminiferous epithelium by immunocytochemistry using monospecific antibodies. Cytochrome cS was lost from the mitochondria as spermatogenesis advanced, while there was a relative increase in cytochrome cT during the zygotene-to-pachytene transition; this was in agreement with other studies that have suggested activation of the cytochrome cT gene during prophase of the first meiotic division. Cytochrome cT was highly concentrated in mitochondria that were being degraded within cytoplasmic lobes of spermatids and in residual bodies that were phagocytized by Sertoli cells. The two isoforms were found to coexist within the same mitochondrion during the transitional period from cytochrome cS to cytochrome cT predominance. In addition, both cytochromes c were present in the chromatoid bodies of spermatocytes and round spermatids; this suggests that the chromatoid body may be involved in the storage of these isozymes and possibly in their differential expression within germ cell mitochondria. Apocytochrome c was concentrated in mitochondria and chromatoid bodies of the germ cells and also scattered in the cytoplasm. The presence of the holoprotein and apoprotein immunoprobes within the chromatoid bodies of spermatocytes and spermatids was an interesting observation that raises questions regarding the precise location of the synthesis of cytochromes c in spermatogenic cells.

Expression of recombinant cytochromes c from various species in Saccharomyces cerevisiae: post-translational modifications.

A complete protocol for the expression of recombinant cytochrome c genes from yeast, Drosophila melanogaster, and rat in a yeast strain, GM-3C-2, which does not express its own cytochromes c is described. The construction of the expression vectors, transformation and large-scale growth of the yeast, and preparation and purification of the recombinant cytochromes c are described. It was found that, contrary to the way yeast modifies its own cytochromes c, the recombinant proteins were partially acetylated at their N-terminus, except for the drosophila protein, which remained entirely unblocked. Furthermore, the yeast and rat proteins were close to fully trimethylated at lysine 72, while the drosophila protein could be separated chromatographically into forms containing tri-, di-, mono-, and unmethylated lysine 72 showing corresponding resonances in the NMR spectrum. These observations emphasize that, in employing expression procedures to obtain native or mutant forms of cytochrome c, it is essential to identify the variety and extent of post-translational modifications and to separate the preparation into pure monomolecular species. Otherwise, it may become impossible to distinguish between the influence of a site-directed mutation and unexamined post-translational modifications.

Relationship between local and global stabilities of proteins: site-directed mutants and chemically-modified derivatives of cytochrome c.

The methionine 80 sulfur-heme iron bond of rat cytochrome c, whose stability is decreased by mutating the phylogenetically invariant residue proline 30 to alanine and increased when tyrosine 67 is changed to phenylalanine, recovers its wild-type characteristics when both substitutions are performed on the same molecule. Titrations with urea, analyzed according to the heteropolymer theory [Alonso, D. O. V., & Dill, K. A. (1991) Biochemistry 30, 5974-5985], indicate that both single mutations increase the solvent exposure of hydrophobic groups in the unfolded state, while in the double mutant this conformational perturbation disappears. Similar increases in solvent exposure of hydrophobic groups are observed when the sulfur-iron bond of the wild-type protein is broken by alkylation of the methionine sulfur, by high pH, or by binding the heme iron with cyanide. The compensatory effects of the two single mutations do not extend to the overall stability of the protein. The added loss of conformational stability due to the single mutations amounts to 7.3 kcal/mol out of the 9 kcal/mol representing the overall free energy of stabilization of the native conformation of the wild-type protein. The folded conformation of the doubly mutated protein is only 2 kcal/mol less stable than that of the wild type. These results indicate that the double mutant protein is able to retain the essential folding pattern of cytochrome c and the thermodynamic stability of the methionine sulfur-heme iron bond, in spite of structural differences that weaken the overall stability of the molecule.