Abstract 1893 Formylation facilitates the reduction of oxidized initiator methionines

Within a cell, protein-bound methionines can be oxidized by reactive oxygen species (ROS) or monooxygenases, and subsequently reduced by methionine sulfoxide reductases (Msrs). Methionine oxidation can result in structural damage or be the basis of functional regulation of enzymes. In addition to participating in redox reactions, methionines also play an important role as the initial residue of translated proteins where they are commonly modified at their α-amine group by formylation or acetylation. Here, we used mass spectrometry-based proteomics to investigate how formylation and acetylation of initiator methionines impact their propensity for oxidation and reduction. We show that within the proteome, N-terminal methionine residues are particularly prone to chemical oxidation, and that their modification by formylation or acetylation greatly enhances their subsequent enzymatic reduction by the two major classes of Msrs in E.coli (MsrA and MsrB). Concordantly, in vivo ablation of methionyl-tRNA formyltransferase (MTF), the enzyme responsible for formylation of methionines, makes E.coli more susceptible to oxidative stress and increases the prevalence of oxidized methionines within translated proteins. We show that oxidation of formylated initiator methionine is detrimental in part because it obstructs their ensuing de-formylation by peptide deformylase (PDF) and hydrolysis by methionyl aminopeptidase (MAP). Thus, by facilitating their reduction, formylation and acetylation mitigate the misprocessing of oxidized initiator methionines and may prevent the misfolding of nascent polypeptides. This work was supported by grants from the National Institutes of Health (R35 GM119502 and S10 OD025242) to S.G. and the Arnold and Mabel Beckman Foundation (Beckman Scholars Program) to M.H.