Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Nitric Oxide. Author manuscript; available in PMC 2017 August 8.
Published in final edited form as:
PMCID: PMC5548592

Nitrosoamphetamine binding to myoglobin and hemoglobin: Crystal structure of the H64A myoglobin-nitrosoamphetamine adduct


N-hydroxyamphetamine (AmphNHOH) is an oxidative metabolite of amphetamine and methamphetamine. It is known to form inhibitory complexes upon binding to heme proteins. However, its interactions with myoglobin (Mb) and hemoglobin (Hb) have not been reported. We demonstrate that the reactions of AmphNHOH with ferric Mb and Hb generate the respective heme-nitrosoamphetamine derivatives characterized by UV-vis spectroscopy. We have determined the X-ray crystal structure of the H64A Mb-nitrosoamphetamine complex to 1.73 Å resolution. The structure reveals the N-binding of the nitroso-d-amphetamine isomer, with no significant H-bonding interactions between the ligand and the distal pocket amino acid residues.

Keywords: phenylhydroxylamine, nitrosoamphetamine, amphetamine, myoglobin, hemoglobin, X-ray structure

1. Introduction

Some commonly prescribed medications contain amphetamine or methamphetamine or a form of either. For example, Adderall®, a drug used to treat attention deficit hyperactivity disorder (ADHD) and narcolepsy, is a combination of amphetamine (Amph; left of Fig. 1) and dextroamphetamine (d-Amph). Desoxyn® is a prescription methamphetamine (methAmph) for the treatment of obesity and ADHD. Despite their high medicinal value, Amph derivatives are known to have high potential for abuse [1]. Because of this, amphetamine and methamphetamine are classified by the U.S. Drug Enforcement Agency as Schedule II substances [1].

Figure 1
Chemical structures of amphetamine (Amph), N-hydroxyamphetamine (AmphNHOH) and N-nitrosoamphetamine (AmphNO).

Interactions of Amph and methAmph with heme proteins are an important component of their metabolism. For example, Amph is oxidatively metabolized by cytochrome P450 to N-hydroxyamphetamine (AmphNHOH; middle of Fig. 1) and other products [2]. In addition, in humans, methAmph is oxidatively metabolized by cytochrome P450 via aromatic hydroxylation and/or N-demethylation to generate Amph and AmphNHOH [3]. It has been known for decades that both Amph and AmphNHOH react with cytochrome P450 under oxidative metabolism conditions to form inhibitory “455 nm” complexes that are stable in the presence of dithionite but are destroyed by ferricyanide [48]. These “455 nm” complexes have been characterized as the nitrosoamphetamine (right of Fig. 1) derivatives containing the active site Fe-AmphNO moiety [7, 9], however no three-dimensional structural information is currently available for these derivatives. We note that such inhibitory Fe-AmphNO derivatives have also been reported for inducible nitric oxide synthase [10], prostaglandin H synthase [11], and microperoxidase-8 [12, 13], but with no structural characterization of the nitroso binding mode. It is intriguing to note that the oxidative metabolism of Amph to AmphNHOH is not limited to cytochrome P450 activity, as human flavin-containing monooxygenase form 3 (FMO3) has been shown to perform this oxidative metabolism as well [14]. Interestingly, FMO3 has also been located in the human brain where it is hypothesized to perform these same functions [15, 16].

AmphNHOH has been isolated as a metabolic intermediate of Amph in animals [17] and surprisingly, formation of heme-AmphNO derivatives have not yet been reported for the muscle protein myoglobin (Mb) and the blood protein hemoglobin (Hb). It has been suggested previously [12, 13] that Mb- and Hb-AmphNO derivatives are unlikely to form due to the large size of AmphNO and the smaller-size heme distal pocket cavity in these proteins.

In this paper, we report the formation of ferrous Mb-AmphNO and Hb-AmphNO derivatives generated from the reactions of AmphNHOH with the ferric heme precursors. Further, we report the 1.73 Å resolution X-ray crystal structure of one such derivative that definitely establishes the coordination mode of the AmphNO ligand to the heme center. The latter structure also represents the first structural evidence for AmphNO binding to any heme protein.

2. Materials and Methods

2.1. Expression and purification of sw Mbs, and isolation of human Hb

The recombinant wt Mb plasmid was a kind gift from Dr. Mario Rivera (Univ. of Kansas). The wt and H64A mutant were expressed in E. coli BL21(DE3) and purified as described by Springer and Sligar [18].

Human blood was obtained from the Blood Bank at the University of Oklahoma Health Science Center in the form of packed cells. OxyHb was isolated from the packed cells and purified following published methods [19, 20]. OxyHb was oxidized to metHb by adding 3 to 5-fold potassium ferricyanide as reported previously [19].

2.2. Crystallization of the sw metMb H64A mutant

Crystals of the recombinant H64A metMb protein were grown as described by Phillips et al [21, 22]. The batch method was applied for crystallization, in which 10–20 μL of 60 mg/ml protein was mixed with 3.2 M ammonium sulfate in 100 mM Tris•HCl, 1 mM EDTA, pH 9.0 to reach 2.3–2.6 M ammonium sulfate as the final concentration.

2.3. Reactions of the heme proteins with N-hydroxyamphetamine

N-hydroxyamphetamine was synthesized following published protocol [23]. The purity was checked by 1H NMR spectroscopy, and the final product was dissolved in methanol for use in the UV-vis studies.

UV-vis spectroscopy was used to monitor the room temperature (22±1 °C) aerobic reactions of the heme proteins with N-hydroxyamphetamine. To establish the starting point of the reaction involving metMb, 10 μL of the protein (40 mg/mL) was added to 2.5 mL of 0.1 M sodium phosphate buffer at pH 7.4 in a quartz cuvette (Starna cells). 10 μL of N-hydroxyamphetamine (90 mM) was then added to the solution. Readings were taken at different time points along the course of the reaction to monitor its progress. Similar procedures were used for the other heme proteins as displayed in the figure captions. The extents of formation of (i.e., conversion to) the products were determined by spectral deconvolution using the program OriginPro (OriginLab, Northampton, MA).

To prepare the H64A Mb derivative in crystalline form, appropriately sized crystals of sw H64A metMb were transferred into a 4 μL droplet (well solution containing 10% glycerol) on a cover slide. The cover slide with the drop was placed in a layer of light mineral oil. A few grains of solid N-hydroxyamphetamine were added into the droplet and the color slowly changed from red to pink signifying the formation of Mb H64A-AmphNO. After a soaking time of 2 d, the treated crystals were harvested and stored in liquid nitrogen until X-ray diffraction data collection.

2.4. X-ray data collection

The diffraction data were collected at our home source using a Rigaku RU-H3R X-ray generator coupled to a R-AXIS IV++ detector. The data were collected at 100 K with CuKα radiation (λ = 1.54178 Å) from the generator operated at 50 kV/100 mA. 1° oscillation angles were collected over a 100° range with an exposure time of 5 min per image and a crystal-to-detector distance of 110 mm.

2.5 Data processing, structure solution and refinement

The data were processed using iMosfilm and SCALA as implemented in the CCP4 program suite [24]. The structure factors were calculated using the CCP4 program suite [25]. The phase problem was solved by molecular replacement using PHASER (CCP4) [26]. The starting model used was wt swMb (metMb-H2O) at 1.5 Å resolution (PDB accession code: 2MBW) with the heme, water molecules, and ligands removed from the structure. All refinements were performed using Refmac5 (CCP4) [27, 28]. Models were rebuilt using COOT [29]. MolProbity was used for structure validation to check for unusual residue conformations contacts [30].

Ten initial cycles of restrained refinement were run with Refmac5, and the R factor decreased from 0.355 to 0.275. Ligands (AmphNO) and water were added to the model based on the Fo-Fc electron density maps in the subsequent refinement cycles. The model of AmphNO comes from the X-ray crystal structure of the heme model compound (OEP)Fe(ONCH(CH3)CH2Ph)(1-MeIm)) (unpublished; OEP = dianion of octaethylporphyrin). To perform restrained refinement in Refmac5, the atoms of AmphNO were selected from this compound in Pymol and saved as another pdb file which was input into Prodrg (CCP4 suite) to generate the coordinates in a new pdb file and the topologies in a monomer library cif file. Two conformations for each of the sidechains of Lys87, Lys133, Lys145 and Gly153 were modeled with 50% occupancy each. The N-terminal Met residue was omitted because of the lack of electron density. The final R factor and Rfree were 0.149 and 0.177, respectively.

The figures were generated using Pymol [31]. 2Fo-Fc electron density maps (e.g., Fig. S6) were initially calculated by Fast Fourier Transform (FFT) in the CCP4 software package [32]. The resulting map files were converted to CCP4 files and displayed in Pymol. In general, each Fo-Fc electron density map was generated as follows: (i) a new PDB file in which ligands were removed from the pocket was initially refined for 5 cycles in Refmac5; (ii) the new mtz file that was generated from the first step was input into FFT to generate the map that was then displayed in Pymol.

3. Results and Discussion

Addition of excess N-hydroxyamphetamine to an aerobic solution of ferric wild-type sperm whale Mb (wt sw metMb) in phosphate buffer at pH 7.4 resulted in spectral changes in the UV-vis spectrum (Fig. 2A). The Soret band of the precursor wt metMb shifted slowly from λmax 409 nm to 423 nm over a 24 hr period (Fig. S1A), and new bands at 543 and 576 nm were also evident. These new spectral bands are indicative of slow (~22%; Fig. S2) conversion of the metMb precursor to the ferrous Mb-nitrosoalkane derivative over this period; other Mb(RNO) complexes display absorption bands in these regions [2, 33]. Similar UV-vis spectral shifts were observed when excess N-hydroxyamphetamine was added to an aerobic solution of the metMb H64A mutant in phosphate buffer at pH 7.4; the Soret band shifted from 408 nm to 426 nm, and new bands at 541 and 573 were evident (Fig. S3). In the case of this H64A mutant, a ~66% conversion to the ferrous Mb-nitrosoalkane derivative was observed after 40 min (Figs. S1B and S4).

Figure 2
UV-vis spectral changes during the aerobic reactions of wt sw metMb (A) and metHb (B) with N-hydroxyamphetamine. Conditions: 0.1 M phosphate buffer, pH 7.4, [Mb] = 2.0–2.5 μM or [Hb] = 12.5 μM, final [reagent] = 80–100 ...

The aerobic reaction of excess N-hydroxyamphetamine with human tetrameric metHb in phosphate buffer at pH 7.4 results in analogous spectral shifts as shown in Fig. 2b; the Soret band shifts from 406 nm to 421 nm, and new bands at 541 and 559 nm appear, with a ~77% conversion occurring over a 4 h period (Figs. S5 and S6).

Once formed, these ferrous nitrosoalkane derivatives are fairly stable in aerobic solution, showing only mild decompostion over time for the Mb products (~12% decomposition over 7 d for wt Mb, ~14% decomposition over 16 d for H64A Mb) and no visible decomposition for the Hb derivative even after 3 d as judged by UV-vis spectroscopy. That these derivatives are in the ferrous form is evidenced by the fact that ferricyanide oxidation coverts them back to their respective ferric precursors (e.g., Fig. S7).

To properly characterize the identity of the ferrous products, we attempted to obtain X-ray diffraction quality crystals for all three derivatives. We have, to date, not been successful at obtaining crystals of any of these products via the co-crystallization method (i.e., crystallization from solution after the product is formed). However, we were successful at obtaining crystals of only the ferrous H64A derivative via the crystal soaking method, where N-hydroxyamphetamine is added to crystals of the H64A metMb precursor (Fig. S8). Specifically, a few grains of solid N-hydroxyamphetamine were added to crystals of H64A metMb in aerobic buffer (0.1 M Tris·HCl, 1 mM EDTA, pH 9.0, 2.3 mM (NH4)2SO4). The crystals changed color from red to pink over a 1 d soaking period. We found, however, that the best condition to obtain diffraction quality crystals of the product was if the soaking was performed over 2 d.

The crystal structure of the ferrous H64A Mb product in the P6 space group was obtained at 1.73 Å resolution (Table S1). The heme active site of this H64A Mb-nitrosoalkane derivative is shown in Fig. 3, and the FoFc omit electron density map shown in the green mesh clearly reveals the presence of the AmphNO ligand (Fig. 1, right) bound to heme Fe.

Figure 3
The FoFc omit electron density map (green mesh, contoured at 3σ) and final model of the heme active site of the ferrous sw H64A Mb-nitrosoamphetamine derivative (PDB accession code 5KD1). The 2FoFc map is shown in Fig. S9. In ...

There are several points to note regarding this H64A Mb-AmphNO structure: (i) the nitrosoamphetamine ligand is bound to heme Fe through its nitroso N atom, (ii) only one isomer, namely the d-Amph isomer, is bound to the Fe center, (iii) the Fe-N-O moiety (FeNO = 108°) is perpendicular to the heme plane and essentially eclipses a heme pyrrole N atom, and (iv) there are no H-bonding interactions between the nitrosoamphetamine ligand and the distal pocket residues. Conformation changes in distal pocket amino acid side-chains when compared with the metMb H64A precursor (rmsd of 0.27Å for main chain Cα atoms, and 0.67 Å for all atoms) are shown in Fig. S10; the Val68 side-chain flips to a “vertical” orientation from its original horizontal position in the metMb H64A precursor, and the Cα atom of the Phe46 residue shifts by 1.5 Å to accommodate the ligand.

The crystal structure of the H64A Mb-AmphNO derivative, and the similarity of its UV-vis spectrum with those of wt Mb and Hb suggest that similar N-binding modes are extant in both Mb and Hb. Importantly, our results show for the first time that bulky amphetamine metabolites such as the nitroso derivative can bind to the relatively small heme sites both Mb and Hb. The formation of heme-AmphNO complexes with Mb and Hb from AmphNHOH suggests that the interactions of AmphNHOH/AmphNO with heme proteins may not be limited to those with large active site pockets such as the cytochrome P450s.

Supplementary Material

Supporting Information


We are grateful to the National Science Foundation (Grants CHE-1213674 and CHE-1566509 to GBR-A) for funding for this work. This paper reports data obtained in the University of Oklahoma Macromolecular Crystallography Laboratory, which is supported, in part, by an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103640, and by a Major Research Instrumentation award from the National Science Foundation under award number 092269. We also thank Dr. Jun (Eva) Yi for her assistance with hemoglobin isolation and purification protocols, and Dr. Paul Sims for assistance with the spectral deconvolution analysis.




Supporting Information: Additional UV-vis spectra; crystallographic data and additional figures (PDF)

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. BW (protein expression, crystallization, crystal soaking, structure solution/refinement, UV-vis), SMP (wtMb /Hb UV-vis), GY (ligand synthesis, H64A UV-vis, crystal soaking), NX (ligand synthesis), GBRA (project coordination). Taken in part from the Ph.D. thesis of GY.


1. U. S. Department of Justice: Drug Enforcement Agency. A DEA Resource Guide. 2015:9, 46, 50–51.
2. Lee J, Chen L, West AH, Richter-Addo GB. Interactions of Organic Nitroso Compounds with Metals. Chem Rev. 2002;102:1019–1065. [PubMed]
3. Li L, Everhart T, Jacob P, III, Jones R, Mendelson J. Stereoselectivity in the Human Metabolism of Metamphetamine. Br J Clin Pharmacol. 2010;69:187–192. [PMC free article] [PubMed]
4. Franklin MR. Complexes of Metabolites of Amphetamines with Hepatic Cytochrome P-450. Xenobiotica. 1974;4:133–142.
5. Franklin MR. The Formation of a 455 nm Complex During Cytochrome P-450-Dependent N-Hydroxyamphetamine Metabolism. Mol Pharmacol. 1974;10:975–985.
6. Jonsson J, Lindeke B. On the Formation of Cytochrome P-450 Product Complexes During the Metabolism of Phenylalkylamines. Acta Pharm Suec. 1976;13:313–320. [PubMed]
7. Mansuy D, Rouer E, Bacot C, Gans P, Chottard JC, Leroux JP. Interaction of Aliphatic N-Hydroxylamines with Microsomal Cytochrome P450: Nature of The Different Derived Complexes and Inhibitory Effects on Monooxygenases Activities. Biochem Pharmacol. 1978;27:1229–1237. [PubMed]
8. James RC, Franklin MR. Comparisons of the Formation of Cytochrome P-450 Complexes Absorbing at 455 nm In Rabbit and Rat Microsomes. Biochem Pharmacol. 1975;24:835–838. [PubMed]
9. Mansuy D, Beaune P, Chottard JC, Bartoli JF, Gans P. The Nature of the “455 nm Absorbing Complex” Formed During the Cytochrome P450 Dependent Oxidative Metabolism of Amphetamine. Biochem Pharmacol. 1976;25:609–612. [PubMed]
10. Renodon A, Boucher JL, Wu C, Gachhui R, Sari MA, Mansuy D, Stuehr D. Formation of Nitric Oxide Synthase-Iron(II) Nitrosoalkane Complexes: Severe Restriction of Access to the Iron(II) Site in the Presence of Tetrahydrobiopterin. Biochemistry. 1998;37:6367–6374. [PubMed]
11. Mahy JP, Mansuy D. Formation of Prostaglandin Synthase-Iron-Nitrosoalkane Inhibitory Complexes upon in situ Oxidation of N-Substituted Hydroxylamines. Biochemistry. 1991;30:4165–4172. [PubMed]
12. Ricoux R, Boucher JL, Mansuy D, Mahy JP. Formation of Iron(II) Nitrosoalkane Complexes: A New Activity of Microperoxidase 8. Biochem Biophys Res Commun. 2000;278:217–223. [PubMed]
13. Ricoux R, Ludowska E, Pezzotti F, Mahy JP. New Activities of a Catalytic Antibody with a Peroxidase Activity: Formation of Fe(II)-RNO Complexes and Stereoselective Oxidation of Sulfides. Eur J Biochem. 2004;271:1277–1283. [PubMed]
14. Cashman JR, Xiong YN, Xu L, Janowsky A. N-Oxygenation of Amphetamine and Methamphetamine by the Human Flavin-Containing Monooxygenase (Form 3): Role in Bioactivation and Detoxification. J Pharmacol Exp Ther. 1999;288:1251–1260. [PubMed]
15. Cashman JR, Zhang J. Human Flavin-Containing Monooxygenases. Ann Rev Pharmacol Toxicol. 2006;46:65–100. [PubMed]
16. Zhang J, Cashman JR. Quantitative Analysis of FMO Gene mRNA Levels in Human Tissues. Drug Metabol Disp. 2006;34:19–26. [PubMed]
17. Beckett AH, Belanger PM. Metabolic Incorporation of Oxygen into Primary and Secondary Aliphatic-Amines and Consequences in Carbon-Nitrogen Bond-Cleavage. J Pharm Pharmacol. 1975;27:547–552. [PubMed]
18. Springer BA, Sligar SG. High-Level Expression of Sperm Whale Myoglobin in Escherichia coli. Proc Natl Acad Sci USA. 1987;84:8961–8965. [PubMed]
19. Antonini E, Brunori M. Hemoglobin and Myoglobin In Their Reactions With Ligands. North-Holland: 1971.
20. Safo MK, Abraham DJ. X-ray Crystallography of Hemoglobins. Methods Mol Med. 2003;82:1–19. [PubMed]
21. Phillips GN, Arduini RM, Springer BA, Sligar SG. Crystal-Structure of Myoglobin from a Synthetic Gene, Proteins. Struc Func Genet. 1990;7:358–365. [PubMed]
22. Smith RD. PhD Thesis. Rice University; Houson, TX: 1999. Correlations Between Bound N-alkyl Isocyanide Orientations and Pathways for Ligand Binding in Recombinant Myoglobins.
23. Mourad MS, Varma RS, Kabalka GW. Reduction of α,β-Unsaturated Nitro-Compounds with Boron Hydrides - a New Route to N-Substituted Hydroxylamines. J Org Chem. 1985;50:133–135.
24. Otwinowski Z, Minor W. Processing of X-Ray Diffraction Data Collected in Oscillation Mode. Methods Enzymol. 1997;276:307–326. [PubMed]
25. Winn MD, Ballard CC, Cowtan KD, Dodson EJ, Emsley P, Evans PR, Keegan RM, Krissinel EB, Leslie AGW, McCoy A, McNicholas SJ, Murshudov GN, Pannu NS, Potterton EA, Powell HR, Read RJ, Vagin A, Wilson KS. Overview of the CCP4 Suite and Current Developments. Acta Crystallogr. 2011;D67:235–242. [PMC free article] [PubMed]
26. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ. PHASER Crystallographic Software. J Appl Cryst. 2007;40:658–674. [PubMed]
27. Murshudov GN, Vagin AA, Dodson EJ. Refinement of Macromolecular Structures by the Maximum-Likelihood Method. Acta Cryst. 1997;D53:240–255. [PubMed]
28. Afonine PV, Grosse-Kunstleve RW, Echols N, Headd JJ, Moriarty NW, Mustyakimov M, Terwilliger TC, Urzhumtsev A, Zwart PH, Adams PD. Towards Automated Crystallographic Structure Refinement with Phenix.refine. Acta Crystallogr. 2012;D68:352–367. [PMC free article] [PubMed]
29. Emsley P, Cowtan K. COOT: Model-Building Tools for Molecular Graphics. Acta Cryst D. 2004;D60:2126–2132. [PubMed]
30. Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, Murray LW, Richardson JS, Richardson DC. MolProbity: All-Atom Structure Validation for Macromolecular Crystallography. Acta Cryst. 2010;D66:12–21. [PMC free article] [PubMed]
31. DeLano WL. The PyMOL Molecular Graphics System. DeLano Scientific LLC; San Carlos, CA, U.S.A: 2006.
32. Read RJ, Schierbeek AJ. A Phased Translation Function. J Appl Crystallogr. 1988;21:490–495.
33. Mansuy D, Chottard JC, Chottard G. Nitrosoalkanes as Fe(II) Ligands in the Hemoglobin and Myoglobin Complexes Formed from Nitroalkanes in Reducing Conditions. Eur J Biochem. 1977;76:617–623. [PubMed]