Insights into Nitrosoalkane Binding to Myoglobin Provided by Crystallography of Wild-Type and Distal Pocket Mutant Derivatives

Abstract

Nitrosoalkanes (R-N═O; R = alkyl) are biological intermediates that form from the oxidative metabolism of various amine (RNH₂) drugs or from the reduction of nitroorganics (RNO₂). 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-H₂O 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-H₂O 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.

Figure 1.

Oxidative conversion of amines (left) and reductive conversion of nitroorganics (right) to organic nitroso compounds and subsequent adduct formation with heme.

Figure 2.

Monodentate binding of RNO to heme centers

Figure 3.

Sketches of the wt and H64A Mb active sites, and the nitrosoalkanes (boxed area) used in this study.

Figure 4.

UV-vis spectral characterization of the reduction of ferric wt sw Mb^III-H₂O by dithionite, followed by the reaction of the resulting ferrous wt sw deoxyMb^II with the nitroalkane (RNO₂) precursors to form the respective ferrous wt sw Mb^II–RNO adducts. A) wt sw Mb^II–MeNO, B) wt sw Mb^II–EtNO, C) wt sw Mb^II–PrNO, and D) wt sw Mb^II–iPrNO. Final reaction conditions: 3 μM wt sw Mb, 0.1 M phosphate buffer (pH 7.4), 20 mM dithionite, 20 mM RNO₂.

Figure 5.

The extent of formation for each wt sw Mb^II–RNO complex as determined by plotting the difference between the absorbances at λ_max 424 and λ 460 nm as a function of time. Absorbance at λ_max 424 nm is indicative of the sw Mb^II–RNO complex and the absorbance at λ 460 nm corresponds to the apparent isosbestic point.

Figure 6.

UV-vis spectral characterization of the reduction of ferric H64A sw Mb^II–H₂O by dithionite, followed by the reaction of ferrous H64A sw deoxyMb^II with the nitroalkane precursors to form the respective ferrous H64A sw Mb^II–RNO adducts. A) H64A sw Mb^II–MeNO, B) H64A sw Mb^II–EtNO, C) H64A sw Mb^II–PrNO, and D) H64A sw Mb^II–iPrNO. Final reaction conditions: 3 μM H64A sw Mb, 0.1 M phosphate buffer (pH 7.4), 20 mM dithionite, 20 mM RNO₂.

Figure 7.

The extent of formation for each ferrous H64A sw Mb^II–RNO complex as determined by plotting the difference between absorbance at λ_max 424 and λ 460 nm as a function of time. The absorbance at λ_max 424 nm is indicative of ferrous H64A sw Mb^II–RNO complex and absorbance at λ 460 nm corresponds to the apparent isosbestic point.

Figure 8.

UV-vis spectra showing conversion of the ferrous wt sw Mb^II–RNO complexes to their ferric wt sw Mb^III–H₂O precursors upon oxidation by ferricyanide. A) wt sw Mb^II–MeNO, B) wt sw Mb^II–EtNO, C) wt sw Mb^II–PrNO, D) wt sw Mb^II–iPrNO. The ferrous wt sw Mb^II–RNO derivatives were prepared as described in the text, followed by the removal of excess dithionite using a desalting column. Afterwards, an aliquot of the sample was placed in a cuvette containing 3 mL of 0.1 M phosphate buffer (pH 6.0), followed by the addition of 3–6 μL of 30 mg/mL potassium ferricyanide.

Figure 9.

Shapes and colors of the crystals obtained in this study by the soaking (s) and co-crystallization (c) methods. A) A brown hexagonal-shaped crystal of ferric sw Mb^III–H₂O indexed with the P6 space group. B) Representative morphology of the violet-pink, ferrous sw Mb^II–RNO hexagonal-shaped crystals indexed with the P6 space group (H64A Mb^II–iPrNO shown here). C) Representative violet-pink thin plate crystals of ferrous sw Mb^II–RNO obtained by the cocrystallization method and indexed with the P12₁1 space group (H64A Mb^II–MeNO shown here).

Figure 10.

Final active site models of wt sw Mb^II bound to (A) MeNO, (B) EtNO, and (C) PrNO. The left panels represent the 2F_o–F_c electron density maps (blue mesh) contoured at 1σ. The right panels show the F_o–F_c omit electron density maps (green mesh) contoured at 3σ. Heme-bound ligands are at 100% occupancy. The MeNO/CH₂=NOH molecule located in the Xe1 pocket of the sw Mb^II–MeNO structure is modeled at 50% occupancy.

Figure 11.

Final active site models of H64A sw Mb^II bound to (A) MeNO, (B) EtNO (Top: Chain A, Bottom: Chain B), (C) PrNO. (D) i-PrNO. The left panels represent the 2F_o–F_c electron density maps (blue mesh) contoured at 1σ. The right panels show the F_o–F_c omit electron density maps (green mesh) contoured at 3σ. Most RNOs are heme bound at 100% occupancy; the exception is EtNO in Chain B, which is bound at 60% occupancy.

Figure 12.

Comparison of the active sites of wt and H64A sw Mb^II–RNO structures by alignment along the Cα chain. A) Cyan wt sw Mb^II–MeNO, gray H64A sw Mb^II–MeNO; B) magenta wt sw Mb^II–EtNO, yellow wt sw Mb^II–PrNO; C) orange H64A sw Mb^II–EtNO Chain A, green H64A sw Mb^II–EtNO Chain B; D) blue H64A sw Mb^II–PrNO, wheat H64A sw Mb^II–iPrNO. The related alignments by pairwise fitting of the 24-atoms of the heme planes are shown in Figure S3 in the Supporting Information.

Figure 13.

Left: (A, left) Superimposed models comparing EtNO binding in wt and H64A sw Mb^II–EtNO in Chain A. (B & C, left) Surface representation showing the vicinity of EtNO in (B, left) wt Mb^II–EtNO and (C, left) H64A Mb^II–EtNO in Chain A. Right: (A, right) Superimposed models comparing PrNO binding in wt and H64A sw Mb. Surface representation showing the vicinity of PrNO in (B, right) wt Mb^II–PrNO and (C, right) H64A Mb^II–PrNO. In both panels, the surface of His/Ala64 and Arg45 are omitted for clarity of view. The black mesh represents the F_o–F_c omit electron density maps contoured at 3σ. Each figure is slightly rotated to highlight the different cavities occupied by the alkyl groups. Alignments shown here were made along the Cα chain.

Figure 14.

Molecular structure of the (PPDME)Fe(iPrNO)(5-MeIm) structure. Thermal ellipsoids are drawn at 40% probability. Hydrogen atoms are not shown for clarity.