Pyruvate dehydrogenase operates as an intramolecular nitroxyl generator during macrophage metabolic reprogramming

M1 macrophages “commit” to a glycolytic state when endogenous nitric oxide (NO) reprograms mitochondrial metabolism by limiting aconitase (Aco)-2 and pyruvate dehydrogenase (PDH). Here we provide evidence that NO directly targets the PDH complex using lipoate to generate nitroxyl (HNO). PDH E2-associated lipoate is modified in NO-rich macrophages while the PDH E3 subunit is irreversibly inhibited. Mechanistically, we show that lipoate facilitates NO-mediated HNO production that interacts with thiols forming modifications resistant to reduction. Consequently, we reveal a macrophage signature of proteins with reduction resistant modifications, including E3, and identify a subset of potential HNO targets. Consistently, recombinant E3 is modified in HNO- dependent manner at Cys⁴⁷⁷ and Cys⁴⁸⁴ , and molecular modeling shows these cause impairment of functional E3 homodimer formation. Our work demonstrates that HNO is produced physiologically, and its presence is peculiar to the lipoate rich PDH complex facilitating irreversible modifications critical in NO-dependent metabolic rewiring.