Cytochrome c is an oxidative stress–activated plasmalogenase that cleaves plasmenylcholine and plasmenylethanolamine at the sn-1 vinyl ether linkage

Plasmalogens are phospholipids critical for cell function and signaling that contain a vinyl ether linkage at the sn-1 position and are highly enriched in arachidonic acid (AA) at the sn-2 position. However, the enzyme(s) responsible for the cleavage of the vinyl ether linkage in plasmalogens has remained elusive. Herein, we report that cytochrome c, in the presence of either cardiolipin (CL), O 2 and H 2 O 2 , or oxidized CL and O 2 , catalyzes the oxidation of the plasmalogen vinyl ether linkage, promoting its hydrolytic cleavage and resultant production of 2-AA-lysolipids and highly reactive -hydroxy fatty aldehydes. Using stable isotope labeling in synergy with strategic chemical derivatizations and high-mass-accuracy MS, we deduced the chemical mechanism underlying this long sought-after reaction. Specifically, labeling with either 18 O 2 or H 2 18 O, but not with H 2 18 O 2 , resulted in M 2 isotopologues of the -hydroxyaldehyde, whereas reactions with both 18 O 2 and H 2 18 O identified the M 4 isotopologue. Furthermore, incorporation of 18 O from 18 O 2 was predominantly located at the -carbon. In contrast, reactions with H 2 18 O yielded 18 O linked to the aldehyde carbon. Importantly, no significant labeling of 2-AA-lysolipids with 18 O 2 , H 2 18 O, or H 2 18 O 2 was present. Intriguingly, phosphatidylinositol phosphates (PIP 2 and PIP 3 ) effectively substituted for cardiolipin. Moreover, cytochrome c released from myocardial mitochondria subjected to oxidative stress cleaved plasmenylcholine in membrane bilayers, and this was blocked with a specific mAb against cytochrome c. Collectively, these results identify the first plasmalogenase in biology, reveal the production of previously unanticipated signaling lipids by cytochrome c, and present new perspectives on cellular signaling during oxidative stress.