Abstract
The catalytic activity of cytochrome c (cyt c) to peroxidize cardiolipin to its oxidized form is required for the release of pro-apoptotic factors from mitochondria, and for execution of the subsequent apoptotic steps. However, the structural basis for this peroxidation reaction remains unclear. In this paper, we determined the three-dimensional NMR solution structure of yeast cyt c Y67H variant with high peroxidase activity, which is almost similar to that of its native form. The structure reveals that the hydrogen bond between Met80 and residue 67 is disrupted. This change destabilizes the sixth coordination bond between heme Fe3+ ion and Met80 sulfur atom in the Y67H variant, and further makes it more easily be broken at low pH conditions. The steady-state studies indicate that the Y67H variant has the highest peroxidase activities when pH condition is between 4.0 and 5.2. Finally, a mechanism is suggested for the peroxidation of cardiolipin catalyzed by the Y67H variant, where the residue His67 acts as a distal histidine, its protonation facilitates O-O bond cleavage of H2O2 by functioning as an acidic catalyst.
Highlights
It has been proposed that proteins can inherently possess a variety of conformations in vivo, and have functional diversity [1]
The cleavage of Fe-S bond generates a vacancy for H2O2 coordination, so that heme ring is easier to be oxidized by H2O2
Mutation from Tyr67 to His67 changes hydrogen bond network in heme pocket To investigate the conformational changes in heme moiety relative to heme ring produced by the mutation from Tyr67 to His67, in Figure 7, the superimpositions were performed by overlaying backbone Ca atoms in secondary structural regions and heme ring including all carbon and nitrogen atoms to form the pconjugate porphyrin system
Summary
It has been proposed that proteins can inherently possess a variety of conformations in vivo, and have functional diversity [1]. Cytochrome c (i.e., cyt c), a well-known electron transfer hemoprotein, has several conformers to carry out its biological functions beyond respiration [4]. The structural investigation on the alkaline conformer implied that this conformer might work as an electron transfer gate, and a folding intermediate [7]. The other is the ‘‘pro-apoptotic’’ conformer with enhanced peroxidase activity [8,9,10]. It can catalyze peroxidation of cardiolipin (a mitochondria specific phospholid) to its oxidized form, which is essential for the release of pro-apoptotic factors from mitochondria, and for performing the subsequent apoptotic processes. The structural basis of this catalytic peroxidation remains unclear
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