Cytochrome c as a Peroxidase: Activation of the Precatalytic Native State by HO-Induced Covalent Modifications.

In addition to serving as respiratory electron shuttle, ferri-cytochrome c (cyt c) acts as a peroxidase; i.e., it catalyzes the oxidation of organic substrates by HO. This peroxidase function plays a key role during apoptosis. Typical peroxidases have a five-coordinate heme with a vacant distal coordination site that permits the iron center to interact with HO. In contrast, native cyt c is six-coordinate, as the distal coordination site is occupied by Met80. It thus seems counterintuitive that native cyt c would exhibit peroxidase activity. The current work scrutinizes the origin of this structure-function mismatch. Cyt c-catalyzed peroxidase reactions show an initial lag phase that is consistent with the in situ conversion of a precatalyst to an active peroxidase. Using mass spectrometry, we demonstrate the occurrence of cyt c self-oxidation in the presence of HO. The newly generated oxidized proteoforms are shown to possess significantly enhanced peroxidase activity. HO-induced modifications commence with oxidation of Tyr67, followed by permanent displacement of Met80 from the heme iron. The actual peroxidase activation step corresponds to subsequent side chain carbonylation, likely at Lys72/73. The Tyr67-oxidized/carbonylated protein has a vacant distal ligation site, and it represents the true peroxidase-active structure of cyt c. Subsequent self-oxidation eventually causes deactivation. It appears that this is the first report that identifies HO-induced covalent modifications as an essential component for the peroxidase activity of "native" cyt c.