Structure and assembly of the diiron cofactor in the heme-oxygenase-like domain of the N-nitrosourea-producing enzyme SznF.

In biosynthesis of the pancreatic cancer drug streptozotocin, the tridomain nonheme-iron oxygenase SznF hydroxylates Nδ and Nω' of Nω-methyl-l-arginine before oxidatively rearranging the triply modified guanidine to the N-methyl-N-nitrosourea pharmacophore. A previously published structure visualized the monoiron cofactor in the enzyme's C-terminal cupin domain, which promotes 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. We leveraged our recent observation that the N-oxygenating µ-peroxodiiron(III/III) intermediate can form in the HO-like domain after the apo protein self-assembles its diiron(II/II) cofactor to solve structures of SznF with both of its iron cofactors bound. These structures of a biochemically validated member of the emerging heme-oxygenase-like diiron oxidase and oxygenase (HDO) superfamily with intact diiron cofactor reveal both the large-scale conformational change required to assemble the O2-reactive Fe2(II/II) complex and the structural basis for cofactor instability-a trait shared by the other validated HDOs. During cofactor (dis)assembly, a ligand-harboring core helix dynamically (un)folds. The diiron cofactor also coordinates an unanticipated Glu ligand contributed by an auxiliary helix implicated in substrate binding by docking and molecular dynamics simulations. The additional carboxylate ligand is conserved in another N-oxygenating HDO but not in two HDOs that cleave carbon-hydrogen and carbon-carbon bonds to install olefins. Among ∼9,600 sequences identified bioinformatically as members of the emerging HDO superfamily, ∼25% conserve this additional carboxylate residue and are thus tentatively assigned as N-oxygenases.

Nonheme-iron histochemistry for light and electron microscopy: a historical, theoretical and technical review.

We reviewed the methods of nonheme-iron histochemistry with special focus on the underlying chemical principles. The term nonheme-iron includes heterogeneous species of iron complexes where iron is more loosely bound to low-molecular weight organic bases and proteins than that of heme (iron-protoporphyrin complex). Nonheme-iron is liberated in dilute acid solutions and available for conventional histochemistry by the Perls and Turnbull and other methods using iron chelators, which depend on the production of insoluble iron compounds. Treatment with strong oxidative agents is required for the liberation of heme-iron, which therefore is not stained by conventional histochemistry. The Perls method most commonly used in laboratory investigations largely stains ferric iron, but stains some ferrous iron as well, while the Turnbull method is specific for the latter. Although the Turnbull method performed on sections fails in staining ferrous iron or stains only such parts of the tissue where iron is heavily accumulated, an in vivo perfusion-Turnbull method demonstrated the ubiquitous distribution of ferrous iron, particularly in lysosomes. The Perls or Turnbull reaction is enhanced by DAB/silver/gold methods for electron microscopy. The iron sulfide method and the staining of redox-active iron with H(2)O(2) and DAB are also applicable for electron microscopy. Although the above histochemical methods have advantages for visualizing iron by conventional light and electron microscopy, the quantitative estimation of iron is not easy. Recent methods depending on the quenching of fluorescent divalent metal indicators by Fe(2+) and dequenching by divalent metal chelators have enabled the quantitative estimation of chelatable Fe(2+) in isolated viable cells.