Mechanism of NO-induced oxidation of myoglobin and hemoglobin
Abstract
Nitric oxide (NO) has been implicated as mediator in a variety of physiological functions, including neurotransmission, platelet aggregation, macrophage function, and vasodilation. The consumption of NO by extracellular hemoglobin and subsequent vasoconstriction have been suggested to be the cause of the mild hypertensive events reported during in vivo trials of hemoglobin-based O2 carriers. The depletion of NO from endothelial cells is most likely due to the oxidative reaction of NO with oxyhemoglobin in arterioles and surrounding tissue. In order to determine the mechanism of this key reaction, we have measured the kinetics of NO-induced oxidation of a variety of different recombinant sperm whale myoglobins (Mb) and human hemoglobins (Hb). The observed rates depend linearly on [NO] but show no dependence on [O2]. The bimolecular rate constants for NO-induced oxidation of MbO2 and HbO2 are large (k.ox,NO = 30-50 microM-1 s-1 for the wild-type proteins) and similar to those for simple nitric oxide binding to deoxygenated Mb and Hb. Both reversible NO binding and NO-induced oxidation occur in two steps: (1) bimolecular entry of nitric oxide into the distal portion of the heme pocket and (2) rapid reaction of noncovalently bound nitric oxide with the iron atom to produce Fe(2+)-N=O or with Fe(2+)-O-O delta- to produce Fe(3+)-OH2 and nitrate. Both the oxidation and binding rate constants for sperm whale Mb were increased when His(E7) was replaced by aliphatic residues. These mutants lack polar interactions in the distal pocket which normally hinder NO entry into the protein. Decreasing the volume of the distal pocket by replacing Leu(B10) and Val(E11) with aromatic amino acids markedly inhibits NO-induced oxidation of MbO2. The latter results provide a protein engineering strategy for reducing hypertensive events caused by extracellular hemoglobin-based O2 carriers. This approach has been explored by examining the effects of Phe(B10) and Phe(E11) substitutions on the rates of NO-induced oxidation of the alpha and beta subunits in recombinant human hemoglobin.
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
NO produced in the red blood cell would be rapidly scavenged by the dioxygenation reaction where NO reacts with oxygenated Hb to form MetHb and nitrate.
“The biggest conceptual challenge to the notion that deoxyHb plays a major role in bioactivation of nitrite is that NO produced in the red blood cell would be rapidly scavenged by the dioxygenation reaction where NO reacts with oxygenated Hb (OxyHb) to form MetHb and nitrate,”
From Mechanisms of nitrite bioactivation (2014) · cites this paper as [43]
In blood, NO reacts with oxygenated hemoglobin to yield nitrate and methemoglobin.
“In blood, NO reacts with oxygenated hemoglobin to yield nitrate and methemoglobin [77].”
From Inorganic nitrite therapy: historical perspective and future directions (2011) · cites this paper as [77]
Cites
PCR protocols — A guide to methods and applications
(1990) · Trends in Genetics · doi:10.1016/0168-9525(90)90186-a · Cited by 11,826 · see citing works
PCR protocols — A guide to methods and applications
U.G. (1990) · Trends in Biochemical Sciences · doi:10.1016/0968-0004(90)90252-7 · Cited by 11,750 · see citing works
The reaction of NO with superoxide
Huie, R. E., & Padmaja, S. (1993) · Free Radical Research Communications · doi:10.3109/10715769309145868 · Cited by 2,121 · see citing works
Nitric oxide synthase structure and mechanism
Marletta, M. A. (1993) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(18)31375-9 · Cited by 1,341 · see citing works
Biosynthesis And Metabolism Of Endothelium-Derived Nitric Oxide
Ignarro, L. J. (1990) · The Annual Review of Pharmacology and Toxicology · doi:10.1146/annurev.pa.30.040190.002535 · Cited by 1,300 · see citing works
Mechanisms of Ligand Recognition in Myoglobin
Springer, B. A., Sligar, S. G., Olson, J. S., & Phillips, G. N. J. (1994) · Chemical Reviews · doi:10.1021/cr00027a007 · Cited by 755 · see citing works
Self-citationOxidation of nitrogen-oxides by bound dioxygen in hemoproteins
Doyle, M. P., & Hoekstra, J. W. (1981) · Journal of Inorganic Biochemistry · doi:10.1016/s0162-0134(00)80291-3 · Cited by 643 · see citing works
High-Resolution Crystal Structures of Distal Histidine Mutants of Sperm Whale Myoglobin
Quillin, M. L., Arduini, R. M., Olson, J. S., & Phillips, G. N. (1993) · Journal of Molecular Biology · doi:10.1006/jmbi.1993.1569 · Cited by 385 · see citing works
Self-citationDiscrimination between Oxygen and Carbon Monoxide and Inhibition of Autooxidation by Myoglobin
Springer, B. A., Egeberg, K. D., Sligar, S. G., Rohlfs, R. J., Mathews, A. J., & Olson, J. S. (1989) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(18)94029-9 · Cited by 288 · see citing works
Self-citationRohlfs, R. J., Mathews, A. J., Carver, T. E., et al. (1990) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(19)39749-2 · Cited by 288 · see citing works
Self-citationKinetics of nitric oxide autoxidation in aqueous solution.
Kharitonov, V. G., Sundquist, A. R., & Sharma, V. (1994) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(17)37543-9 · Cited by 224 · see citing works
Carver, T. E., BRANTLEY, R., Singleton, E. W., et al. (1992) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(19)49732-9 · Cited by 211 · see citing works
His64(E7)–>Tyr apomyoglobin as a reagent for measuring rates of hemin dissociation.
Hargrove, M. S., Singleton, E. W., Quillin, M. L., Ortiz, L. A., Phillips, G. N., Olson, J. S., & Mathews, A. J. (1994) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(17)41764-9 · Cited by 210 · see citing works
Self-citationStructural and Functional Effects of Apolar Mutations of the Distal Valine in Myoglobin
Quillin, M. L., Li, T., Olson, J. S., Phillips, G. N., Jr., Duo, Y., Ikeda‐Saito, M., Regan, R. M., Carlson, M. L., Gibson, Q. H., Li, H., et al. (1995) · Journal of Molecular Biology · doi:10.1006/jmbi.1994.0034 · Cited by 190 · see citing works
Self-citationStructure of myoglobin-ethyl isocyanide histidine as a swinging door for ligand entry
Johnson, K. A., Olson, J. S., & Phillips, G. N. (1989) · Journal of Molecular Biology · doi:10.1016/0022-2836(89)90269-6 · Cited by 126 · see citing works
Self-citationCrystal structure of myoglobin form a synthetic gene
Phillips, G. N., Arduini, R. M., Springer, B. A., et al. (1990) · Proteins Structure Function and Bioinformatics · doi:10.1002/prot.340070407 · Cited by 110 · see citing works
Blood substitutes : physiological basis of efficacy
Winslow, R. M., Vandegriff, K. D., & Intaglietta, M. (1995) · Medical Entomology and Zoology · Cited by 109 · see citing works
Structural factors governing azide and cyanide binding to mammalian metmyoglobins.
Brancaccio, A., Cutruzzolà, F., Allocatelli, C. T., Brunori, M., Smerdon, S. J., Wilkinson, A. J., Dou, Y., Keenan, D., Ikeda‐Saito, M., & BRANTLEY, R. (1994) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(17)36724-8 · Cited by 108 · see citing works
Egeberg, K. D., Springer, B. A., Sligar, S. G., et al. (1990) · Journal of Biological Chemistry · doi:10.1016/s0021-9258(19)38467-4 · Cited by 103 · see citing works
ESR spectral transition by arteriovenous cycle in nitric oxide hemoglobin of cytokine-treated rats
Kosaka, H., Sawai, Y., Sakaguchi, H., Kumura, E., Harada, N., Watanabe, M., & Shiga, T. (1994) · American Journal of Physiology-Cell Physiology · doi:10.1152/ajpcell.1994.266.5.c1400 · Cited by 95 · see citing works
Physiological factors affecting O2 transport by hemoglobin in an in vitro capillary system
Lemon, D. D., Nair, P. K., Boland, E. J., et al. (1987) · Journal of Applied Physiology · doi:10.1152/jappl.1987.62.2.798 · Cited by 52 · see citing works
Self-citationBlood Substitutes: Engineering the Haemoglobin Molecule
Vandegriff, K. D. (1992) · Biotechnology and Genetic Engineering Reviews · doi:10.1080/02648725.1992.10647894 · Cited by 28 · see citing works
Flagged first, then by citations
Cited by
Sorted by citations
Cell-free hemoglobin limits nitric oxide bioavailability in sickle-cell disease
Reiter, C. D., Wang, X., Tanus-Santos, J. E., Hogg, N., Cannon, R. O., Schechter, A. N., & Gladwin, M. T. (2002) · Nature Medicine · doi:10.1038/nm1202-799 · Cited by 1,330 · see citing works
Thomas, D. D., Liu, X., Kantrow, S. P., & Lancaster, J. R. (2001) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.98.1.355 · Cited by 760 · see citing works
Nitric oxide metabolism and breakdown
Kelm, M. (1999) · Biochimica et Biophysica Acta (BBA) - Bioenergetics · doi:10.1016/s0005-2728(99)00020-1 · Cited by 673 · see citing works
Strategies to increase nitric oxide signalling in cardiovascular disease
Lundberg, J. O., Gladwin, M. T., & Weitzberg, E. (2015) · Nature Reviews Drug Discovery · doi:10.1038/nrd4623 · Cited by 576 · see citing works
Sources of Vascular Nitric Oxide and Reactive Oxygen Species and Their Regulation
Tejero, J., Shiva, S., & Gladwin, M. T. (2019) · Physiological Reviews · doi:10.1152/physrev.00036.2017 · Cited by 541 · see citing works
Nitric oxide and iron proteins
Cooper, C. E. (1999) · Biochimica et Biophysica Acta (BBA) - Bioenergetics · doi:10.1016/s0005-2728(99)00021-3 · Cited by 510 · see citing works
Diffusion-limited reaction of free nitric oxide with erythrocytes
Liu, X., Miller, M. J. S., Joshi, M. S., Sadowska-Krowicka, H., Clark, D. A., & Lancaster, J. R. (1998) · Journal of Biological Chemistry · doi:10.1074/jbc.273.30.18709 · Cited by 512 · see citing works
Nitrite as Regulator of Hypoxic Signaling in Mammalian Physiology
van Faassen, E. E., Bahrami, S., Feelisch, M., Hogg, N., Kelm, M., Kim‐Shapiro, D. B., Kozlov, A. V., Li, H., Lundberg, J. O., Mason, R., et al. (2009) · Medicinal Research Reviews · doi:10.1002/med.20151 · Cited by 435 · see citing works
Maia, L. B., & Moura, J. J. G. (2014) · Chemical Reviews · doi:10.1021/cr400518y · Cited by 401 · see citing works
What is the real physiological NO concentration in vivo?
Hall, C. N., & Garthwaite, J. (2009) · Nitric Oxide · doi:10.1016/j.niox.2009.07.002 · Cited by 378 · see citing works
Nagababu, E., Ramasamy, S., Abernethy, D. R., & Rifkind, J. M. (2003) · Journal of Biological Chemistry · doi:10.1074/jbc.m307572200 · Cited by 349 · see citing works
Unraveling the Reactions of Nitric Oxide, Nitrite, and Hemoglobin in Physiology and Therapeutics
Kim-Shapiro, D. B., Schechter, A. N., & Gladwin, M. T. (2006) · Arteriosclerosis, Thrombosis, and Vascular Biology · doi:10.1161/01.atv.0000204350.44226.9a · Cited by 308 · see citing works
Endothelial cell expression of haemoglobin α regulates nitric oxide signalling
Straub, A. C., Lohman, A. W., Billaud, M., Johnstone, S. R., Dwyer, S. T., Lee, M. Y., Bortz, P. S., Best, A. K., Columbus, L., Gaston, B., et al. (2012) · Nature · doi:10.1038/nature11626 · Cited by 308 · see citing works
Kinetic pathways and barriers for ligand binding to myoglobin
Olson, J. S., & Phillips, G. N. (1996) · Journal of Biological Chemistry · doi:10.1074/jbc.271.30.17593 · Cited by 259 · see citing works
Kapil, V., Khambata, R. S., Jones, D. A., Rathod, K., Primus, C., Massimo, G., Fukuto, J. M., & Ahluwalia, A. (2020) · Pharmacological Reviews · doi:10.1124/pr.120.019240 · Cited by 248 · see citing works
Catalytic generation of N2O3 by the concerted nitrite reductase and anhydrase activity of hemoglobin
Basu, S., Grubina, R., Huang, J., Conradie, J., Huang, Z., Jeffers, A., Jiang, A., He, X., Azarov, I., Seibert, R., et al. (2007) · Nature Chemical Biology · doi:10.1038/nchembio.2007.46 · Cited by 224 · see citing works
Joshi, M. S., Ferguson, T. B., Han, T. H., Hyduke, D. R., Liao, J. C., Rassaf, T., Bryan, N., Feelisch, M., & Lancaster, J. R. (2002) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.152149699 · Cited by 216 · see citing works
Gladwin, M. T., Grubina, R., & Doyle, M. P. (2009) · Accounts of Chemical Research · doi:10.1021/ar800089j · Cited by 194 · see citing works
The Redox Activity of Hemoglobins: From Physiologic Functions to Pathologic Mechanisms
Reeder, B. J. (2010) · Antioxidants & Redox Signaling · doi:10.1089/ars.2009.2974 · Cited by 189 · see citing works
Mechanisms of nitrite bioactivation
Kim-Shapiro, D. B., & Gladwin, M. T. (2014) · Nitric Oxide · doi:10.1016/j.niox.2013.11.002 · Cited by 157 · see citing works
Hemoglobin-mediated nitric oxide signaling
Helms, C., & Kim-Shapiro, D. B. (2013) · Free Radical Biology and Medicine · doi:10.1016/j.freeradbiomed.2013.04.028 · Cited by 146 · see citing works
Diet-induced endogenous formation of nitroso compounds in the GI tract
Kuhnle, G. G. C., Story, G. W., Reda, T., Mani, A. R., Moore, K. P., Lunn, J. C., & Bingham, S. A. (2007) · Free Radical Biology and Medicine · doi:10.1016/j.freeradbiomed.2007.03.011 · Cited by 146 · see citing works
Nitrite Reduction and Cardiovascular Protection
Omar, S. A., & Webb, A. J. (2014) · Journal of Molecular and Cellular Cardiology · doi:10.1016/j.yjmcc.2014.01.012 · Cited by 121 · see citing works
Inorganic nitrite therapy: historical perspective and future directions
Kevil, C. G., Kolluru, G. K., Pattillo, C. B., & Giordano, T. (2011) · Free Radical Biology and Medicine · doi:10.1016/j.freeradbiomed.2011.04.042 · Cited by 113 · see citing works
Platelet Inhibition by Nitrite Is Dependent on Erythrocytes and Deoxygenation
Srihirun, S., Sriwantana, T., Unchern, S., Kittikool, D., Noulsri, E., Pattanapanyasat, K., Fucharoen, S., Piknova, B., Schechter, A. N., & Sibmooh, N. (2012) · PLoS ONE · doi:10.1371/journal.pone.0030380 · Cited by 109 · see citing works
Hemoglobin-Mediated, Hypoxia-Induced Vasodilation via Nitric Oxide
Robinson, J. M., & Lancaster, J. R. (2005) · American Journal of Respiratory Cell and Molecular Biology · doi:10.1165/rcmb.f292 · Cited by 104 · see citing works
Mitochondria as metabolizers and targets of nitrite
Shiva, S. (2010) · Nitric Oxide · doi:10.1016/j.niox.2009.09.002 · Cited by 87 · see citing works
Álvares, T. S., Conte, C. A., Paschoalin, V. M. F., Silva, J. T., Meirelles, C. D. M., Bhambhani, Y. N., & Gomes, P. S. C. (2012) · Applied Physiology, Nutrition, and Metabolism · doi:10.1139/h11-144 · Cited by 71 · see citing works
The potential role of the red blood cell in nitrite-dependent regulation of blood flow
Patel, R. P., Hogg, N., & Kim-Shapiro, D. B. (2011) · Cardiovascular Research · doi:10.1093/cvr/cvq323 · Cited by 70 · see citing works
Nitric oxide red blood cell membrane permeability at high and low oxygen tension
Huang, K. T., Huang, Z., & Kim-Shapiro, D. B. (2007) · Nitric Oxide · doi:10.1016/j.niox.2006.11.002 · Cited by 62 · see citing works
Nitrosation, thiols, and hemoglobin: energetics and kinetics
Koppenol, W. H. (2012) · Inorganic Chemistry · doi:10.1021/ic202561f · Cited by 54 · see citing works
Reeder, B. J., & Ukeri, J. (2018) · Nitric Oxide · doi:10.1016/j.niox.2017.11.004 · Cited by 50 · see citing works
Comparing the chemical biology of NO and HNO
Flores-Santana, W., Switzer, C., Ridnour, L. A., Basudhar, D., Mancardi, D., Donzelli, S., Thomas, D. D., Miranda, K. M., Fukuto, J. M., & Wink, D. A. (2009) · Archives of Pharmacal Research · doi:10.1007/s12272-009-1805-x · Cited by 50 · see citing works
Neuroglobin ligand binding kinetics
Kiger, L., Uzan, J., Dewilde, S., Burmester, T., Hankeln, T., Moens, L., Hamdane, D., Baudin‐Creuza, V., & Marden, M. C. (2004) · IUBMB Life · doi:10.1080/15216540500037711 · Cited by 48 · see citing works
Hendgen-Cotta, U. B., Kelm, M., & Rassaf, T. (2010) · Nitric Oxide · doi:10.1016/j.niox.2009.10.003 · Cited by 46 · see citing works
Blood, A. B., Schroeder, H. J., Terry, M. H., Merrill-Henry, J., Bragg, S. L., Vrancken, K., Liu, T., Herring, J. L., Sowers, L. C., Wilson, S. M., et al. (2011) · Circulation · doi:10.1161/circulationaha.110.001073 · Cited by 36 · see citing works
Red Blood Cell Membrane-Facilitated Release of Nitrite-Derived Nitric Oxide Bioactivity
Salgado, M. T., Cao, Z., Nagababu, E., Mohanty, J. G., & Rifkind, J. M. (2015) · Biochemistry · doi:10.1021/acs.biochem.5b00643 · Cited by 34 · see citing works
The emerging roles of somatic globins in cardiovascular redox biology and beyond
Rahaman, M. M., & Straub, A. C. (2013) · Redox Biology · doi:10.1016/j.redox.2013.08.001 · Cited by 24 · see citing works
“Although this provides a mechanism to limit NO toxicity, the reaction creates a physiological paradox, whereby abundant concentrations of Hb in red blood cells ( 10 mM) should reduce NO bioavailability from endothelium to vascular smooth muscle.”
BackgroundRifkind, J. M., Nagababu, E., Cao, Z., Barbiro-Michaely, E., & Mayevsky, A. (2009) · Advances in experimental medicine and biology · doi:10.1007/978-0-387-85998-9_5 · Cited by 10 · see citing works
- Authors
- Eich, R. F., Li, T., Lemon, D. D., Doherty, D. H., Curry, S. R., Aitken, J. F., Mathews, A. J., Johnson, K. A., Smith, R. D., Phillips, & Olson, J. S.
- Affiliations
- Rice University (US)
- Department of Biochemistry and Cell Biology and the W. M. Keck Center for Computational Biology, Rice University, 6100 Main Street, Houston, Texas 77005-1892, and Somatogen, Inc., 2545 Central Avenue, Boulder, Colorado 80301
- Corresponding author
- Olson, John S.
- Venue
- Biochemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 1996-01-01
- Volume
- 35
- Issue
- 22
- Pages
- 6976-6983
- ISSN
- 0006-2960 (ISSN-L)
- 1520-4995
- OpenAlex
- W1974936925
- Cited by
- 613
- References
- 22
- Citations by year
- 2026: 4 · 2025: 7 · 2024: 10 · 2023: 8 · 2022: 19 · 2021: 11 · 2020: 19 · 2019: 12 · 2018: 15 · 2017: 22 · 2016: 14 · 2015: 17 · 2014: 17 · 2013: 34 · 2012: 29
- FWCI
- 6.83
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Biochemistry (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institutes of Health (7r01ar040252-11)
- National Heart, Lung, and Blood Institute (HL47020, R01HL047020)
- National Institute of General Medical Sciences (GM35649, R01GM035649)
- National Institute of Arthritis and Musculoskeletal and Skin Diseases (AR 40252)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Hemoglobin structure and function
- Physical Sciences › Chemistry › Inorganic Chemistry › Metal-Catalyzed Oxygenation Mechanisms
- Health Sciences › Medicine › Physiology › Nitric Oxide and Endothelin Effects
- Keywords
- nitric oxide, myoglobin, hemoglobin, oxyhemoglobin, sperm whale myoglobin, iron nitrosyl, nitrate formation, protein engineering, hemoglobin-based oxygen carriers, vasoconstriction, hypertension
- MeSH terms
- Animals, Binding Sites, Hemoglobins, Humans, Kinetics, Models, Molecular, Myoglobin, Nitric Oxide, Nitriles, Oxidation-Reduction, Protein Binding, Recombinant Proteins, Whales, Protein Engineering, Mutagenesis, Site-Directed
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review
