Alternative Conformations of Yeast Iso-1-Cytochrome C: Effects of a Gatekeeping Residue on Heme Crevice Dynamics

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We have solved a novel crystal structure of yeast iso-1-cytochrome c to 1.45 A displaying water coordinated to the iron of the heme. This conformation of cytochrome c (Cytc) is capable of the peroxidase activity required for oxidation of cardiolipin by Cytc in the beginning stages of apoptosis. The native Met80 ligand from Ω-loop D has swung away from the heme coordination site with very little backbone rearrangement. Local sterics have been relaxed in Ω-loop D facilitating access to this conformation by replacement of trimethyllysine 72 with alanine. Trimethyllysine lies across the surface of Ω-loop D contacting Thr78, Met80 and Ala81. Ordered waters within the heme crevice form a buried water channel providing an accessible pathway from the exterior of Cytc to the heme which peroxide and protons can move through. Alternate conformations of Arg38, observed in the crystal structure, control access of the water channel to bulk water at the protein surface. Although peroxidase activity is similar near pH 6 for Cytc with trimethyllisine and alanine at position 72, between pH 7 and 8 peroxidase activity is increased 1.5 to 2-fold in the Lys72Ala variant. Loop dynamics from pH jump stopped-flow experiments demonstrate a 2-fold faster rate of opening for Ω-loop D when alanine is present at position 72. Increased Ω-loop D dynamics and peroxidase activity of the Lys72Ala variant suggests that residue 72 acts as a gatekeeper for the opening of Ω-loop D, regulating the ease with which Met80 can swing outwards providing access to the heme coordination site. The Lys72Ala mutation has allowed visualization of a conformer of yeast iso-1-Cytc competent for the peroxidase activity which occurs in the early stages of the intrinsic apoptosis pathway.