Nitroorganics present a general concern for a safe environment due to their health hazards. However, some nitroorganics such as metronidazole (Mtz) and chloramphenicol (CAM) also possess medicinal value. Mtz and CAM can undergo reductive bioactivation presumably via their nitroso derivatives. We show, using UV-vis spectroscopy, that sperm whale myoglobin (swMb) and its distal pocket mutants retaining H-bonding capacity react with Mtz in the presence of dithionite to generate products with spectra suggestive of the Fe-bound nitroso (Fe–RNO; λmax [Formula: see text]420 nm) forms. We have crystallized and solved the X-ray crystal structure of an H64Q swMb-acetamide compound to 1.76 Å resolution; the formation of this compound results from the serendipitous crystallographic trapping, by the heme center, of acetamide from the reductive decomposition of Mtz. Only one of the swMb proteins, namely H64Q swMb with a relatively flexible Gln64 residue, reacted with CAM presumably due to the bulky nature of CAM that generally may restrict its access to the heme site.
Nitrosoalkanes (R−N�O; R = alkyl) are biological intermediates that form from the oxidative metabolism of various amine (RNH 2 ) drugs or from the reduction of nitroorganics (RNO 2 ). RNO compounds bind to and inhibit various heme proteins. However, structural information on the resulting Fe−RNO moieties remains limited. We report the preparation of ferrous wild-type and H64A sw Mb II −RNO derivatives (λ max 424 nm; R = Me, Et, Pr, iPr) from the reactions of Mb III −H 2 O with dithionite and nitroalkanes. The apparent extent of formation of the wt Mb derivatives followed the order MeNO > EtNO > PrNO > iPrNO, whereas the order was the opposite for the H64A derivatives. Ferricyanide oxidation of the Mb II −RNO derivatives resulted in the formation of the ferric Mb III −H 2 O precursors with loss of the RNO ligands. X-ray crystal structures of the wt Mb II −RNO derivatives at 1.76−2.0 Å resoln. revealed N-binding of RNO to Fe and the presence of H-bonding interactions between the nitroso O-atoms and distal pocket His64. The nitroso O-atoms pointed in the general direction of the protein exterior, and the hydrophobic R groups pointed toward the protein interior. X-ray crystal structures for the H64A mutant derivatives were determined at 1.74−1.80 Å resoln. An analysis of the distal pocket amino acid surface landscape provided an explanation for the differences in ligand orientations adopted by the EtNO and PrNO ligands in their wt and H64A structures. Our results provide a good baseline for the structural analysis of RNO binding to heme proteins possessing small distal pockets.
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