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.

中文翻译：

---

N-亚硝基脲生产酶SznF的血红素加氧酶样结构域中二铁辅因子的结构和组装

在胰腺癌药物链脲佐菌素的生物合成中，三结构域非血红素铁加氧酶SznF在将三重修饰的胍氧化为N-甲基-N-亚硝基脲药效团后，将N-甲基-L-精氨酸的两个N原子羟基化。 。先前发布的结构在酶的C末端铜蛋白结构域中可视化了单铁辅因子，这影响了最终的重排，但在拟议的二铁辅因子的N-羟基化血红素加氧酶样位点中表现出无序和最小的金属占有率（类似于HO）的中央域。在这里，我们利用我们最近关于由Fe2（II / II）络合物和O2形成的高吸收性过二铁（III / III）中间体的报告，了解二铁辅因子在HO类结构域中的组装并获得具有SznF铁的结构辅助因子绑定。一个铁离子亚位点的紧密结合与构象变化有关，随后是第二个亚位点的弱结合，Fe2（II / II）络合物快速捕获O2。缺铁和富铁结构之间的差异揭示了形成O2反应性Fe2（II / II）络合物所需的构象变化和辅因子不稳定性的结构基础，表明配体携带的核心螺旋在金属获取过程中动态重折叠然后释放。辅助因子还通过停靠和分子动力学模拟来协调由参与底物结合的辅助螺旋贡献的意外Glu配体。在另一种经过实验验证的HO样N加氧酶中，该额外的配体是保守的，但在两种已知的HO样二铁去饱和酶中却没有。