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.

Mass spectrometric identification of lysine residues of heme oxygenase-1 that are involved in its interaction with NADPH-cytochrome P450 reductase.

The lysine residues of rat heme oxygenase-1 (HO-1) were acetylated by acetic anhydride in the absence and presence of NADPH-cytochrome P450 reductase (CPR) or biliverdin reductase (BVR). Nine acetylated peptides were identified by MALDI-TOF mass spectrometry in the tryptic fragments obtained from HO-1 acetylated without the reductases (referred to as the fully acetylated HO-1). The presence of CPR prevented HO-1 from acetylation of lysine residues, Lys-149 and Lys-153, located in the F-helix. The heme degradation activity of the fully acetylated HO-1 in the NADPH/CPR-supported system was significantly reduced, whereas almost no inactivation was detected in HO-1 in the presence of CPR, which prevented acetylation of Lys-149 and Lys-153. On the other hand, the presence of BVR showed no protective effect on the acetylation of HO-1. The interaction of HO-1 with CPR or BVR is discussed based on the acetylation pattern and on molecular modeling.

Heme oxygenase and NO-synthase in the human prostate--relation to adrenergic, cholinergic and peptide-containing nerves.

In the human prostate, the distribution of heme oxygenase (HO-1 and HO-2)-, nitric oxide synthase (NOS)-, and tyrosine hydroxylase (TH)-immunoreactive (IR), acetylcholine-esterase (AChE)-positive, and some peptidergic nerve structures was investigated. Cell bodies and nerve fibers within coarse nerve trunks expressed HO-1-, HO-2-, NOS-, TH-, and vasoactive intestinal polypeptide (VIP)-immunoreactivities, and were AChE-positive, but, as revealed by confocal microscopy. HO- and NOS-immunoreactivities were found in separate nerves. Along strains of smooth muscle, intraglandular septa, and around acini, HO-1-, NOS-, and VIP-IR nerves, and AChE-positive fibers were observed. Double immunostaining showed that NOS- and VIP-immunoreactivities were generally co-localized in varicose nerve terminals. Some TH-IR terminals had profiles that were similar, but not identical, to those of NOS-, HO-1-, or VIP-IR terminals. NPY-IR nerves were similarly distributed as VIP- and NOS-IR fibers, and were found in rich amounts. Calcitonin gene-related peptide (CGRP)-IR nerves were few compared to other nerve populations studies. NOS- and CGRP-IR terminals had similar profiles, but the immunoreactivities were not co-localized. Nitric oxide and electrical stimulation of nerves relaxed noradrenaline-contracted preparations of prostatic stroma. Inhibition of synthesis of nitric oxide abolished the electrically induced relaxations. VIP had small relaxant effects, whereas carbon monoxide was without effect on noradrenaline-contracted strips. The innervation pattern and the functional effects suggest that the L-arginine/nitric oxide pathway may have a role in the control of human prostatic smooth muscle activity and/or in secretory neurotransmission. A physiological role of carbon monoxide in the prostate remains to be established.