title: Deoxymyoglobin is a nitrite reductase that generates nitric oxide and regulates mitochondrial respiration.
abstract: Previous studies have revealed a novel interaction between deoxyhemoglobin and nitrite to generate nitric oxide (NO) in blood. It has been proposed that nitrite acts as an endocrine reservoir of NO and contributes to hypoxic vasodilation and signaling. Here, we characterize the nitrite reductase activity of deoxymyoglobin, which reduces nitrite approximately 36 times faster than deoxyhemoglobin because of its lower heme redox potential. We hypothesize that physiologically this reaction releases NO in proximity to mitochondria and regulates respiration through cytochrome c oxidase. Spectrophotometric and chemiluminescent measurements show that the deoxymyoglobin-nitrite reaction produces NO in a second order reaction that is dependent on deoxymyoglobin, nitrite and proton concentration, with a bimolecular rate constant of 12.4 mol/L(-1)s(-1) (pH 7.4, 37 degrees C). Because the IC(50) for NO-dependent inhibition of mitochondrial respiration is approximately 100 nmol/L at physiological oxygen tensions (5 to 10 mumol/L); we tested whether the myoglobin-dependent reduction of nitrite could inhibit respiration. Indeed, the addition of deoxymyoglobin and nitrite to isolated rat heart and liver mitochondria resulted in the inhibition of respiration, while myoglobin or nitrite alone had no effect. The addition of nitrite to rat heart homogenate containing both myoglobin and mitochondria resulted in NO generation and inhibition of respiration; these effects were blocked by myoglobin oxidation with ferricyanide but not by the xanthine oxidoreductase inhibitor allopurinol. These data expand on the paradigm that heme-globins conserve and generate NO via nitrite reduction along physiological oxygen gradients, and further demonstrate that NO generation from nitrite reduction can escape heme autocapture to regulate NO-dependent signaling.
authors.affiliation: Vascular Medicine Branch, Clinical Center; National Institutes of Health, Bethesda, MD 20892-1662, USA.
authors.forename: Sruti
authors.initials: S
authors.lastname: Shiva
authors.forename: Zhi
authors.initials: Z
authors.lastname: Huang
authors.forename: Rozalina
authors.initials: R
authors.lastname: Grubina
authors.forename: Junhui
authors.initials: J
authors.lastname: Sun
authors.forename: Lorna A
authors.initials: LA
authors.lastname: Ringwood
authors.forename: Peter H
authors.initials: PH
authors.lastname: MacArthur
authors.forename: Xiuli
authors.initials: X
authors.lastname: Xu
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Murphy
authors.forename: Victor M
authors.initials: VM
authors.lastname: Darley-Usmar
authors.forename: Mark T
authors.initials: MT
authors.lastname: Gladwin
mesh_terms: D000818:Animals; D019069:Cell Respiration / Q000502:physiology; D006418:Heme / Q000378:metabolism; D006736:Horses; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008297:Male; D008929:Mitochondria, Heart / Q000201:enzymology* / Q000378:metabolism; D008930:Mitochondria, Liver / Q000201:enzymology* / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D009211:Myoglobin / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D009569:Nitric Oxide / Q000096:biosynthesis / Q000378:metabolism*; D009572:Nitrite Reductases / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D009573:Nitrites / Q000037:antagonists & inhibitors / Q000378:metabolism; D010084:Oxidation-Reduction; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D009211:Myoglobin; D009573:Nitrites; C055547:deoxymyoglobin; D009569:Nitric Oxide; D006418:Heme; D009572:Nitrite Reductases