cytochrome-c-t and trimethyllysine

Trimethyllysine 72 (tmK72) has been suggested to play a role in sterically constraining the heme crevice dynamics of yeast iso-1-cytochrome c mediated by the Ω-loop D cooperative substructure (residues 70-85). A tmK72A mutation causes a gain in peroxidase activity, a function of cytochrome c that is important early in apoptosis. More than one higher energy state is accessible for the Ω-loop D substructure via tier 0 dynamics. Two of these are alkaline conformers mediated by Lys73 and Lys79. In the current work, the effect of the tmK72A mutation on the thermodynamic and kinetic properties of wild-type iso-1-cytochrome c (yWT versus WT*) and on variants carrying a K73H mutation (yWT/K73H versus WT*/K73H) is studied. Whereas the tmK72A mutation confers increased peroxidase activity in wild-type yeast iso-1-cytochrome c and increased dynamics for formation of a previously studied His79-heme alkaline conformer, the tmK72A mutation speeds return of the His73-heme alkaline conformer to the native state through destabilization of the His73-heme alkaline conformer relative to the native conformer. These opposing behaviors demonstrate that the response of the dynamics of a protein substructure to mutation depends on the nature of the perturbation to the substructure. For a protein substructure which mediates more than one function of a protein through multiple non-native structures, a mutation could change the partitioning between these functions. The current results suggest that the tier 0 dynamics of Ω-loop D that mediates peroxidase activity has similarities to the tier 0 dynamics required to form the His79-heme alkaline conformer.

Topics: Cytochromes c; Hydrogen-Ion Concentration; Kinetics; Lysine; Models, Molecular; Saccharomyces cerevisiae; Sodium Chloride; Thermodynamics

Trimethyllysine 72 (Tml72) of yeast iso-1-cytochrome c lies across the surface of the heme crevice loop (Ω-loop D, residues 70-85) like a brace. Lys72 is oriented similarly in horse cytochrome c (Cytc). To determine whether this residue affects the dynamics of opening the heme crevice loop, we have studied the effect of a Tml72 to Ala substitution on the formation of the His79-heme alkaline conformer near neutral pH using a variant of iso-1-Cytc including K72A and K79H mutations. Guanidine hydrochloride denaturation shows that the Tml72 to Ala substitution within error does not affect the global stability of the protein. The effect of the Tml72 to Ala substitution on the thermodynamics of the His79-heme alkaline transition is also small. However, pH-jump kinetic studies of the His79-heme alkaline transition show that both the forward and backward rates of conformational change are increased by the Tml72 to Ala substitution. The barrier for opening the heme crevice is reduced by 0.5 kcal/mol and for closing the heme crevice by 0.3 kcal/mol. The ability of Tml72 to modulate the heme crevice dynamics may indicate a crucial role in regulating function, such as in the peroxidase activity seen in the early stages of apoptosis.

Topics: Alanine; Amino Acid Substitution; Cytochromes c; Heme; Hydrogen-Ion Concentration; Kinetics; Lysine; Models, Molecular; Protein Conformation; Protein Denaturation; Protein Stability; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Thermodynamics

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.

Topics: Amino Acid Sequence; Animals; Apoptosis; Cytochrome c Group; Cytochromes c; Horses; Lysine; Methylation; Mitochondria; Models, Molecular; Molecular Sequence Data; Oocytes; Peptide Hydrolases; Protein Isoforms; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Sequence Homology, Amino Acid; Xenopus

The reduction of wild-type yeast iso-1-ferricytochrome c (ycytc) and several mutants by trypsin-solubilized bovine liver ferrocytochrome b5 (cytb5) has been studied under conditions in which the electron-transfer reaction is bimolecular. The effect of electrostatic charge modifications and steric changes on the kinetics has been determined by experimental and theoretical observations of the electron-transfer rates of ycytc mutants K79A, K'72A, K79A/K'72A, and R38A (K' is used to signify trimethyllysine (Tml)). A structurally robust Brownian dynamics (BD) method simulating diffusional docking and electron transfer was employed to predict the mutation effect on the rate constants. A realistic model of the electron-transfer event embodied in an intrinsic unimolecular rate constant is used which varies exponentially with donor-acceptor distance. The BD method quantitatively predicts rate constants over a considerable range of ionic strengths. Semiquantitative agreement is obtained in predicting the perturbing influence of the mutations on the rate constants. Both the experimentally observed rate constants and those predicted by BD descend in the following order: native ycytc > K79A > K'72A > K79A/K'72A. Variant R38A was studied at a different ionic strength than this series of mutations, and the theory agreed with experiment in predicting a smaller rate constant for the mutant. In all cases the predicted effect of mutation was in the correct direction, but not as large as that observed. The BD simulations predict that the two proteins dock through essentially a single domain, with a distance of closest approach of the two heme groups in rigid body docking typically around 12 A. Two predominant classes of complexes were calculated, the most frequent involving the quartet of cytb5/ycytc interactions, Glu48-Arg13, Glu56-Lys87, Asp60-Lys86, and heme-Tml72, having an average electrostatic energy of -13.0 kcal/mol. The second most important complexes were of the type previously postulated (Salemme, 1976; Mauk et al., 1986; Rodgers et al., 1988) with interactions Glu44-Lys27, Glu48-Arg13, Asp60-Tml72, and heme-Lys79 and having an energy of -6.4 kcal/mol. The ionic strength dependence of the bimolecular reaction rate was well reproduced using a discontinuous dielectric model, but poorly so for a uniform dielectric model.

Topics: Amino Acid Sequence; Animals; Binding Sites; Cattle; Cytochrome c Group; Cytochromes b5; Cytochromes c; Heme; Histidine; Kinetics; Liver; Lysine; Mutagenesis, Site-Directed; Oxidation-Reduction; Protein Conformation; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins

1. A mutant of the iso-1-cytochrome c gene from Saccharomyces cerevisiae has been constructed which contains an Arg codon, replacing the normal trimethylated Lys at position 77. 2. This mutated gene was cloned into a pGem 1 vector and used for the in vitro translation of yeast iso-1-cytochrome c. 3. Utilizing an in vitro mitochondria binding assay, it was found that the mutant cytochrome c could transverse the yeast mitochondrial membrane, however the amount of protein incorporated was 3-fold less that of the trimethylated wild type. 4. Omission of the protein methyltransferase from assays containing the wild type cytochrome c caused only a slight reduction (15%) in the amount of protein incorporated. 5. These results suggest while the lysine residue 77 of apocytochrome c is important for mitochondria uptake, the methylation of this residue seems to play a relatively minor role.

Topics: Animals; Arginine; Base Sequence; Cloning, Molecular; Codon; Cytochrome c Group; Cytochromes c; Lysine; Methylation; Mitochondria; Mitochondria, Liver; Molecular Sequence Data; Mutagenesis, Site-Directed; Polymerase Chain Reaction; Protein Biosynthesis; Rats; Restriction Mapping; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Structure-Activity Relationship; Transcription, Genetic