At the onset of apoptosis, the peroxidation of cardiolipin at the inner mitochondrial membrane by cytochrome c requires an open coordination site on the heme. We report a 1.45-Å resolution structure of yeast iso-1-cytochrome c with the Met80 heme ligand swung out of the heme crevice and replaced by a water molecule. This conformational change requires modest adjustments to the main chain of the heme crevice loop and is facilitated by a trimethyllysine 72-to-alanine mutation. This mutation also enhances the peroxidase activity of iso-1-cytochrome c. The structure shows a buried water channel capable of facilitating peroxide access to the active site and of moving protons produced during peroxidase activity to the protein surface. Alternate positions of the side chain of Arg38 appear to mediate opening and closing of the buried water channel. In addition, two buried water molecules can adopt alternate positions that change the network of hydrogen bonds in the buried water channel. Taken together, these observations suggest that low and high proton conductivity states may mediate peroxidase function. Comparison of yeast and mammalian cytochrome c sequences, in the context of the steric factors that permit opening of the heme crevice, suggests that higher organisms have evolved to inhibit peroxidase activity, providing a more stringent barrier to the onset of apoptosis.

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The kinetics of the alkaline conformational transition of a Lys 73-->His variant of iso-1-cytochrome c have been investigated using pH jump stopped-flow methods to probe the nature of the ionizable "trigger" group for this conformational change. This mutation moves the pK(a) of the ligand replacing Met 80 from about 10.5 to approximately 6.6 and has unmasked two other ionizable groups, besides the ligand replacing Met 80, that modulate the kinetics of this process. The results are discussed in terms of the impact of ionization equilibria on protein folding mechanisms.

The alkaline transition kinetics of a Lys 73-->His (H73) variant of iso-1-cytochrome c are triggered by three ionizable groups [Martinez, R. E., and Bowler, B. E. (2004) J. Am. Chem. Soc. 126, 6751-6758]. To eliminate ambiguities caused by overlapping phases due to formation of the Lys 79 alkaline conformer and proline isomerization associated with the His 73 alkaline conformer, we mutated Lys 79 to Ala in the H73 variant (A79H73). The stability and guanidineHCl m-values of the A79H73 and H73 variants at pH 7.5 are the same. The Ala 79 mutation causes formation of the alkaline conformer to depend on [NaCl]. The salt dependence saturates at 500 mM NaCl, and the thermodynamics of alkaline state formation for the A79H73 and H73 variants become identical. The salt dependence is consistent with loss of an electrostatic contact between Lys 79 and heme propionate D in the A79H73 variant. The kinetics of alkaline state formation for the A79H73 variant support the three trigger group model developed for the H73 variant, with the primary trigger, pK(HL), being ionization of His 73. The low pH ionization, pK(H1), is perturbed by the Ala 79 mutation indicating that this ionization is modulated by the buried hydrogen bond network involving heme propionate D. The A79H73 variant has a high spin heme above pH 9 suggesting that the high pH ionization, pK(H2), involves a high spin heme conformer. The proline isomerization phase is modulated by both pK(HL) and pK(H2) indicating that it is sensitive to protein conformation.

A series of mutations at the highly solvent-exposed lysine 73 of iso-1-cytochrome c have been prepared by site-directed mutagenesis. These mutations were designed to probe denatured-state effects on the unfolding equilibrium of this protein. The hydrophilic amino acid Lys was replaced with the hydrophobic amino acids Met, Tyr, Phe, and Trp. The idea was to induce stabilizing hydrophobic interactions in the unfolded state, while having little effect on the folded-state energy due to the high solvent exposure of this site. Fourier transform infrared spectral analyses indicate that none of these mutations significantly affect the native fold of the protein. The stability of each protein to guanidine hydrochloride denaturation was monitored at 25 degrees C by circular dichroism spectroscopy. All four hydrophobic mutants decreased the value of delta Go uH2O, the free energy of unfolding of the protein in the absence of denaturant, by 1.0-1.5 kcal/mol. The delta Go uH2O values for these proteins correlate linearly (correlation coefficient of 0.98) with the hydrophobicity of the amino acid at position 73 of the sequence. These data are consistent with the idea that the position-73 mutants are more buried in the denatured state than in the native state, suggestive of a compact denatured state where such interactions would be possible.

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