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Biocoordination chemistry of HNO/NO with Hemes

In the past, any possible biological activity of nitrite oxide, NO, was discounted because of its high reactivity and toxicity. But in the last twenty years, NO has been demonstrated to play many different roles in our bodies: controlling blood pressure, long-term memory and the immune response. The physiological importance of nitric oxide has generated tremendous interest in the chemistry of heme-nitrosyls, as both the formation and activity of NO is directly attributable to heme cofactors in nitric oxide synthase and soluble guanyl cyclase.

There is also considerable interest in the biological activity of the one-electron reduced form of nitric oxide, termed a nitroxyl or nitrosyl hydride (NO- or HNO). Nitroxyl intermediates have been proposed in the catalytic cycles of the heme enzymes nitric oxide synthase (NOS) and the nitrite and nitric oxide reductases (NiR and NoR). The pharmacological activity of nitroxyl-releasing drugs has also been suggested to be due to their reactivity with the various heme targets.


HNO adducts of myoglobin

Metal complexes of HNO are rare and typically short-lived in solution. A notable exception is the HNO adduct of myoglobin, Mb-HNO, which we first observed during an electrochemical study (Bayachou JACS 1998). Subsequently, we synthesized it directly by reduction of Mb-NO (Lin JACS 2000); the nitroxyl was protonated at nitrogen, as demonstrated by the splitting of its 1H NMR at ca. 15 ppm. The unique 1H NMR signal of the HNO adduct allowed us to determine its solution structure by NOE and COSY NMR methods in collaboration with Gerd LaMar at UC Davis (Sulc, JBIC 2003).

HNO itself is very short-lived in aqueous solution due to a nearly diffusion-controlled dimerization, but we have shown that deoxymyoglobin efficiently traps HNO to give Mb-HNO directly (Sulc JACS 2004). The binding of HNO to Mb is rapid and essentially irreversible; this implies that the beneficial effects of nitroxyl for heart disease may be mediated by its reactivity with myoglobin and hemoglobin in the heart and blood. We have also recently collaborated on a re-evaluation of the oxidation potential and pKa of HNO, which overturned some common misconceptions on its possible biological generation (Bartberger PNAS 2002).

More recent work has been on the photolysis of HNO-Mb, which produces a transient geminate pair of HNO- and FeIII Mb (Pervitsky, JACS 2008) and descriptions of HNO trapping by other oxygen binding globins like human hemoglobin, leghemoglobin from soybeans and a hemoglobin from clams. (Kumar, Biochem 2009). In this latter work, we used HSQC to access the 15N chemical shifts of the nitrosyl hydride adducts, and showed that allosteric changes accompany the binding of HNO to tetrameric human hemoglobin. With collaborators, we have also been looking at the effect of HNO – releasing compounds on other oxygen and/or NO binding proteins like soluble guanylate cyclase and tyrosinase. We also believe the coordination chemistry of HNO will be a fruitful area for both metalloprotein and small molecule chemistry.

NMR spectra of the HNO adduct of human hemoglobin in pH 7 iP buffer. At bottom are the 1H NMR spectra A) in the HNO region, B) in the valine region. At top are the corresponding C) 1H-15N HSQC spectrum of H15NO adduct and D) 2D 1H-1H NOESY spectrum showing the interaction between nitrosyl hydride and valine methyl protons.




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Selected publications on N-oxide chemistry:

  • “The effects of nitroxyl (HNO) on soluble guanylate cyclase activity: Interactions at ferrous heme and cysteine thiols" Miller, T.M.; Cherney, M.E.; Franco, N.; Farmer, P.J.; King, S.B.; Hobbs, A.J.; Miranda, K.; Burstyn, J.N.; Fukuto, J.M. J. Biol. Chem. 2009, 284, 21788-21796.
  • “Nitrosyl hydride (HNO) as an O2 analogue: long-lived HNO-adducts of ferrous globins” Kumar, M.R.; Pervitsky, D.; Chen, L.; Poulos, T.L.; Kundu, S.; Hargrove, M.S.; Rivera, E.J.; Colón, J.M.; Farmer, P.J. Biochemistry, 2009, 48, 5018–5025.
  • “Genome-wide siRNAi-based Functional Genomics of Pigmentation Identifies Regulatory Networks Governing Melanogenesis in Human Cells” Ganesan, A.K.: Ho, H.; Bodemann, B.; Petersen, S.; Aruri, J.; Koshy, S.; Richardson, Z.; Le, L.Q.; Krasieva, T.; Roth, M.G.; Farmer, P.J.; White, M.A. PLoS Genetics, 2008 4, e1000298.
  • “Photolysis of the HNO Adduct of Myoglobin: Transient Generation of the Aminoxyl Radical” Pervitsky, D.; Immoos, C.; van der Veer, W.; Farmer, P.J. J. Am. Chem. Soc. 2007, 129, 9590-9591.
  • “The interaction of nitric oxide with distinct hemoglobins differentially amplifies endothelial heme uptake and heme oxygenase-1 expression” Foresti, R.: Bains, S.; Sulc, F.; Farmer, P.J.; Green, C.J.; Motterlini, R. J Pharmacol Exp Ther. 2006, 317, 1125-1133.
  • “Bonding in HNO-Myoglobin as Characterized by X-Ray Absorbance and Resonance Raman Spectroscopies” Immoos, C.E.; Sulc, F.; Farmer, P.J.; Czarnecki, K.; Bocian, D.F.; Levina, A.; Aitken, J.B.; Armstrong, R.S.; Lay, P.A. J. Am. Chem. Soc.; 2005, 127, 814 – 815.
  • “Coordination Chemistry of the HNO Ligand with Hemes and Synthetic Coordination Complexes” Farmer, P.J.; Sulc, F. J. Inorg. Biochem. 2005, 99, 166-184.
  • “Trapping of Nitroxyl by Deoxy Myoglobin” Sulc, F.; Immoos, C.; Pervitsky, D. Farmer, P. J. J. Amer. Chem. Soc. 2004, 125, 1096-1101.
  • "1H NMR Structure of the Heme Pocket of HNO-Myoglobin" Sulc, F.; Fleischer, E.; Farmer, P. J.; Ma, D.; La Mar, G. J. Biol. Inorg. Chem. 2003, 8, 348-352.
  • "The reduction potential of nitric oxide (NO) and its importance to NO biochemistry" Bartberger, M.D.; Liu, W.; Ford, E.; Miranda, K. M.; Switzer, C.; Fukuto, J. M.; Farmer, P. J.; Wink, D.A.; Houk K. N. Proc. Nat. Acad. Sci. 2002, 99, 10958-10963.
  • "O-Atom Transfer from Nitric Oxide Catalyzed by Fe(TPP)" Lin, R.; Farmer, P. J. J. Am. Chem. Soc. 2001, 123, 1143 -1150.
  • “The HNO Adduct of Myoglobin: Synthesis and Characterization” Lin, R.; Farmer, P. J. J. Am. Chem. Soc 2000, 122, 2393 -2394.