Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2020.07.29.227702v1?rss=1 Authors: McBride, M. J., Pope, S. R., Hu, K., Slater, J. W., Okafor, C. D., Balskus, E. P., Bollinger, J. M., Boal, A. K. Abstract: In biosynthesis of the pancreatic cancer drug streptozotocin, the tri-domain nonheme-iron oxygenase, SznF, hydroxylates two N-atoms of N-methyl-L-arginine before oxidatively rearranging the triply modified guanidine to the N-methyl-N-nitrosourea pharmacophore. A previously published structure visualized the mono-iron cofactor in the enzyme's C-terminal cupin domain, which effects the final rearrangement, but exhibited disorder and minimal metal occupancy in the site of the proposed diiron cofactor in the N-hydroxylating heme-oxygenase-like (HO-like) central domain. Here we leverage our recent report of an intensely absorbing peroxodiiron(III/III) intermediate formed from the Fe2(II/II) complex and O2 to understand assembly of the diiron cofactor in the HO-like domain and to obtain structures with both SznF iron cofactors bound. Tight binding at one diiron subsite is associated with a conformational change, which is followed by weak binding at the second subsite and rapid capture of O2 by the Fe2(II/II) complex. Differences between iron-deficient and iron-replete structures reveal both the conformational change required to form the O2-reactive Fe2(II/II) complex and the structural basis for cofactor instability, showing that a ligand-harboring core helix dynamically refolds during metal acquisition and release. The cofactor also coordinates an unanticipated Glu ligand contributed by an auxiliary helix implicated in substrate binding by docking and molecular dynamics simulation. The additional ligand is conserved in another experimentally validated HO-like N-oxygenase but not in two known HO-like diiron desaturases. Among ~9600 sequences identified bioinformatically as belonging to the emerging HO-like diiron protein (HDO) superfamily, ~25% have this carboxylate residue and are thus tentatively assigned as N-oxygenases. Copy rights belong to original authors. Visit the link for more info