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Bioorganometallic Fe–C bonds are biologically relevant species that may result from the metabolism of natural or synthetic hydrazines. The molecular structures of four new sperm whale mutant myoglobin derivatives with Fe–aryl moieties, namely H64A–tolyl- m , H64A–chlorophenyl- p , H64Q–tolyl- m , and H64Q–chlorophenyl- p , have been determined at 1.7–1.9 Å resolution. The structures reveal conformational preferences for the substituted aryls resulting from attachment of the aryl ligands to Fe at the site of net –NHNH 2 release from the precursor hydrazines, and show distal pocket changes that readily accommodate these bulky ligands. [ABSTRACT FROM AUTHOR]

Amphetamine-based (Amph) drugs are metabolized in humans to their hydroxylamine (AmphNHOH) and nitroso (AmphNO) derivatives. The latter metabolites are known to bind to the Fe centers of cytochrome P450 and other heme enzymes to inhibit their activities. Although these AmphNHOH/AmphNO metabolites are present in vivo, their interactions with the blood protein hemoglobin (Hb) and the muscle protein (Mb) have been largely discounted due to a perception that the relatively small heme active sites of Hb and Mb will not be able to accommodate the large AmphNO group. We report the 2.15 Å resolution X-ray crystal structure of the AmphNO adduct of adult human hemoglobin as the Hb α -FeIII(H 2 O)] β -FeII(AmphNO)] derivative. We show that the binding of AmphNO to the β subunit is enabled by an E helix movement and stabilization of ligand binding by H-bonding with the distal His63 residue. We also observe an AmphNHOH group in the Xe2 pocket in close proximity to the α heme site in this derivative. Additionally, UV–vis spectroscopy was used to characterize this and related wt and mutant Mb adducts. Importantly, our X-ray crystal structure of this Hb-nitrosoamphetamine complex represents the first crystal structure of a wild-type heme protein adduct of any amphetamine metabolite. Our results provide a framework for further studies of AmphNHOH/AmphNO interactions with Hb and Mb as viable processes that potentially contribute to the overall biological inorganic chemistry of amphetamine drugs. A nitrosoamphetamine (AmphNO) adduct of human adult hemoglobin (Hb), namely Hb α -FeIII(H 2 O)] β -FeII(AmphNO)], displaying direct binding of an amphetamine metabolite to the β heme Fe center, has been characterized by X-ray crystallography to 2.15 Å resolution. This represents the first structural determination of an amphetamine derivative bound to a wild-type protein. Unlabelled Image • The crystal structure of a nitrosoamphetamine adduct of hemoglobin was determined. • The nitrosoamphetamine ligand is bound to the β -heme Fe via its N-atom. • E-helix movements allow for the binding of this large ligand in the distal pocket. [ABSTRACT FROM AUTHOR]

Phenylhydroxylamine (PhNHOH) and nitrosobenzene (PhNO) interact with human tetrameric hemoglobin (Hb) to form the nitrosobenzene adduct Hb(PhNO). These interactions also frequently lead to methemoglobin formation in red blood cells. We utilize UV–vis spectroscopy and X-ray crystallography to identify the primary and secondary products that form when PhNHOH and related alkylhydroxylamines (RNHOH; R = Me, t -Bu) react with human ferric Hb. We show that with MeNHOH, the primary product is Hb[α-FeIII(H 2 O)][β-FeII(MeNO)], in which nitrosomethane is bound to the β subunit but not the α subunit. Attempts to isolate a nitrosochloramphenicol (CAMNO) adduct resulted in our isolation of a Hb[α-FeII][β-FeII-cySOx] {CAMNO} product (cySOx = oxidized cysteine) in which CAMNO was located outside of the protein in the solvent region between the β2 and α2 subunits of the same tetramer. We also observed that the βcys93 residue had been oxidized. In the case of t -BuNHOH, we demonstrate that the isolated product is the β-hemichrome Hb[α-FeIII(H 2 O)][β-FeIII(His) 2 ] { t -BuNHOH} , in which the β heme has slipped ∼4.4 Å towards the solvent exterior to accommodate the bis-His heme coordination. When PhNHOH is used, a similar β-hemichrome Hb[α-FeIII(H 2 O)][β-FeIII(His) 2 -cySOx] {PhNHOH} was obtained. Our results reveal, for the first time, the X-ray structural determination of a β-hemichrome in a human Hb derivative. Our UV–vis and X-ray crystal structural result reveal that although Hb(PhNO) and Hb(RNO) complexes may form as primary products, attempted isolation of these products by crystallization may result in the structural determination of their secondary products which may contain β-hemichromes en route to further protein degradation. A nitrosomethane (MeNO) adduct of human hemoglobin (Hb), namely Hb[ α -FeIII(H 2 O)][ β -FeII(MeNO)], in which MeNO is bound only to the β-heme has been characterized by X-ray crystallography. In contrast, β-hemichrome structures were obtained in attempts to crystallize the products from the reactions of Hb with t -BuNHOH and PhNHOH. [Display omitted] • The crystal structure of a nitrosomethane adduct of hemoglobin was determined. • Hb β-hemichrome crystal structures have been determined. • Large heme slippages to accommodate β-hemichrome formation were observed. [ABSTRACT FROM AUTHOR]

Staphylococcus aureus (Sa) is a serious concern due to increasing resistance to antibiotics. The bacterial dihydrofolate reductase enzyme is effectively inhibited by trimethoprim, a compound with antibacterial activity. Previously, we reported a trimethoprim derivative containing an acryloyl linker and a dihydophthalazine moiety demonstrating increased potency against S. aureus. We have expanded this series and assessed in vitro enzyme inhibition (K i) and whole cell growth inhibition properties (MIC). Modifications were focused at a chiral carbon within the phthalazine heterocycle, as well as simultaneous modification at positions on the dihydrophthalazine. MIC values increased from 0.0626–0.5 μg/mL into the 0.5–1 μg/mL range when the edge positions were modified with either methyl or methoxy groups. Changes at the chiral carbon affected K i measurements but with little impact on MIC values. Our structural data revealed accommodation of predominantly the S -enantiomer of the inhibitors within the folate-binding pocket. Longer modifications at the chiral carbon, such as p -methylbenzyl, protrude from the pocket into solvent and result in poorer K i values, as do modifications with greater torsional freedom, such as 1-ethylpropyl. The most efficacious K i was 0.7 ± 0.3 nM, obtained with a cyclopropyl derivative containing dimethoxy modifications at the dihydrophthalazine edge. The co-crystal structure revealed an alternative placement of the phthalazine moiety into a shallow surface at the edge of the site that can accommodate either enantiomer of the inhibitor. The current design, therefore, highlights how to engineer specific placement of the inhibitor within this alternative pocket, which in turn maximizes the enzyme inhibitory properties of racemic mixtures. Image 1 • Trimethoprim derivatives offer additional chemical diversity within a proven scaffold. • Altering chemical moieties at the binding site: solvent interface modulates affinity. • Altering chemical moieties at the dihydrophthalazine edge alters cell inhibition. • Combining moieties improves inhibition by targeting a unique binding site surface. • Derivatives targeting this site do not prefer a specific enantiomer. [ABSTRACT FROM AUTHOR]