Structure and dynamics of a muti-domain nitric oxide synthase regulated by a C2 domain

Abstract Nitric oxide synthase (NOS) is a widely studied multidomain redox enzyme that produces the key signaling molecule and cytotoxic agent nitric oxide (NO) for functions that range from mammalian vasodilation to prokaryotic antibiotic resistance. NOS enzymes from metazoans and cyanobacteria rely on dynamic associations of their oxygenase and coupled di-flavin reductase domains that have largely evaded detailed structural characterization. CryoEM studies of a representative dimeric six-domain Synechococcus NOS reveal the architecture of the full-length enzyme, which contains an unusual regulatory C2 domain, and additional nitric oxide deoxygenase (NOD) and pseudo-globin modules. Five distinct structural states depict how pterin binding couples to tight and loose oxygenase conformations and how the Ca2+-sensitive C2 domain moves over 85 Å to alternatively regulate either the NOS or NOD heme center. The extended C-terminal tail and its dynamic interactions highlight an added layer of regulation required by multidomain NOSs compared to other di-flavin reductases. Teaser syNOS is a highly dynamic multi-domain oxidoreductase that harnesses a Ca2+-sensitive C2 domain to modulate activity. Competing Interest Statement The authors have declared no competing interest. Footnotes Revisions to suit peer review. Additional experiments were conducted and portions of the text was cleaned up.