iNOS modulates inflammatory responses in an NO-independent manner through direct interaction with IRG1 in mitochondria

. Here we show that iNOS inhibits IRG1 activity and itaconate levels through a conformation-dependent protein-protein interaction rather than through the production of NO. Using a variety of biochemical and computational approaches, we show that a direct interaction between iNOS and IRG1 occurs within mitochondria, in mouse and human cells, and that it depends on binding of the cofactor BH4 to iNOS but does not require its capability to produce NO. Our findings reveal a non-canonical cellular function for iNOS that places it at the centre of a signalling hub, linking redox signalling and metabolism to modulation of the inflammatory response in macrophages.