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), O2 and H2 O2 , or oxidized CL and O2 , catalyzes the oxidation of the plasmalogen vinyl ether linkage, promoting its hydrolytic cleavage and resultant production of 2-AA-lysolipids and highly reactive α-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 O2 or H2 18 O, but not with H2 18 O2 , resulted in M + 2 isotopologues of the α-hydroxyaldehyde, whereas reactions with both 18 O2 and H2 18 O identified the M + 4 isotopologue. Furthermore, incorporation of 18 O from 18 O2 was predominantly located at the α-carbon. In contrast, reactions with H2 18 O yielded 18 O linked to the aldehyde carbon. Importantly, no significant labeling of 2-AA-lysolipids with 18 O2 , H2 18 O, or H2 18 O2 was present. Intriguingly, phosphatidylinositol phosphates (PIP2 and PIP3 ) 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.