Nitric oxide interactions with cobalamins: biochemical and functional consequences

Abstract Nitric oxide (NO) is a paramagnetic gas that has been implicated in a wide range of biologic functions. The common pathway to evoke the functional response frequently involves the formation of an iron- nitrosyl complex in a target (heme) protein. In this study, we report on the interactions between NO and cobalt-containing vitamin B12 derivatives. Absorption spectroscopy showed that of the four Co(III) derivatives (cyanocobalamin [CN-Cbl], aquocobalamin [H2O-Cbl], adenosylcobalamin [Ado-Cbl], and methylcobalamin [MeCbl]), only the H2O- Cbl combined with NO. In addition, electron paramagnetic resonance spectroscopy of H2O-Cbl preparations showed the presence of a small amount of Cob-(II)alamin that was capable of combining with NO. The Co(III)-NO complex was very stable, but could transfer its NO moiety to hemoglobin (Hb). The transfer was accompanied by a reduction of the Co(III) to Co(II), indicating that NO+ (nitrosonium) was the leaving group. In accordance with this, the NO did not combine with the Hb Fe(II)-heme, but most likely with the Hb cysteine-thiolate. Similarly, the Co(III)-NO complex was capable of transferring its NO to glutathione. Ado-Cbl and Me-Cbl were susceptible to photolysis, but CN- Cbl and H2O-Cbl were not. The homolytic cleavage of the Co(III)-Ado or Co(III)-Me bond resulted in the reduction of the metal. When photolysis was performed in the presence of NO, formation of NO-Co(II) was observed. Co(II)-nitrosyl oxidized slowly to form Co(III)-nitrosyl. The capability of aquocobalamin to combine with NO had functional consequences. We found that nitrosylcobalamin had diminished ability to serve as a cofactor for the enzyme methionine synthase, and that aquocobalamin could quench NO-mediated inhibition of cell proliferation. Our in vitro studies therefore suggest that interactions between NO and cobalamins may have important consequences in vivo.

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Nitric oxide interactions with cobalamins: biochemical and functional consequences

Blood ◽

10.1182/blood.v88.5.1857.bloodjournal8851857 ◽

1996 ◽

Vol 88 (5) ◽

pp. 1857-1864

Author(s):

M Brouwer ◽

W Chamulitrat ◽

G Ferruzzi ◽

DL Sauls ◽

JB Weinberg

Keyword(s):

Nitric Oxide ◽

Electron Paramagnetic Resonance Spectroscopy ◽

Resonance Spectroscopy ◽

Paramagnetic Resonance ◽

No Formation ◽

Iron Nitrosyl ◽

Wide Range ◽

Functional Consequences

Nitric oxide (NO) is a paramagnetic gas that has been implicated in a wide range of biologic functions. The common pathway to evoke the functional response frequently involves the formation of an iron- nitrosyl complex in a target (heme) protein. In this study, we report on the interactions between NO and cobalt-containing vitamin B12 derivatives. Absorption spectroscopy showed that of the four Co(III) derivatives (cyanocobalamin [CN-Cbl], aquocobalamin [H2O-Cbl], adenosylcobalamin [Ado-Cbl], and methylcobalamin [MeCbl]), only the H2O- Cbl combined with NO. In addition, electron paramagnetic resonance spectroscopy of H2O-Cbl preparations showed the presence of a small amount of Cob-(II)alamin that was capable of combining with NO. The Co(III)-NO complex was very stable, but could transfer its NO moiety to hemoglobin (Hb). The transfer was accompanied by a reduction of the Co(III) to Co(II), indicating that NO+ (nitrosonium) was the leaving group. In accordance with this, the NO did not combine with the Hb Fe(II)-heme, but most likely with the Hb cysteine-thiolate. Similarly, the Co(III)-NO complex was capable of transferring its NO to glutathione. Ado-Cbl and Me-Cbl were susceptible to photolysis, but CN- Cbl and H2O-Cbl were not. The homolytic cleavage of the Co(III)-Ado or Co(III)-Me bond resulted in the reduction of the metal. When photolysis was performed in the presence of NO, formation of NO-Co(II) was observed. Co(II)-nitrosyl oxidized slowly to form Co(III)-nitrosyl. The capability of aquocobalamin to combine with NO had functional consequences. We found that nitrosylcobalamin had diminished ability to serve as a cofactor for the enzyme methionine synthase, and that aquocobalamin could quench NO-mediated inhibition of cell proliferation. Our in vitro studies therefore suggest that interactions between NO and cobalamins may have important consequences in vivo.

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Adventitia-derived nitric oxide in rat aortas exposed to endotoxin: cell origin and functional consequences

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.2000.279.6.h2743 ◽

2000 ◽

Vol 279 (6) ◽

pp. H2743-H2751 ◽

Cited By ~ 28

Author(s):

Andrei L. Kleschyov ◽

Bernard Muller ◽

Thérèse Keravis ◽

Marie-Elisabeth Stoeckel ◽

Jean-Claude Stoclet

Keyword(s):

Nitric Oxide ◽

Inflammatory Diseases ◽

Early Stage ◽

Rat Aorta ◽

Paramagnetic Resonance ◽

Dinitrosyl Iron ◽

Functional Consequences ◽

Resonance Spin

The role of adventitial cells in bacterial lipopolysaccharide (LPS)-induced vascular nitric oxide (NO) overproduction has been largely ignored. In rat aortas exposed to LPS in vitro or in vivo, it was found that adventitia contained the major part of NO synthase (NOS)-2 protein (Western blot and immunohistochemistry) and generated the largest amount of NO (electron paramagnetic resonance spin trapping). NOS-2 immunoreactive cells were mainly resident macrophages at an early stage (5 h, in vitro or in vivo) and fibroblasts at a later stage (20 h, in vitro). Adventitial NOS-2 activity largely accounted for 1) the relaxing effect of l-arginine in rings exposed to LPS in vivo, 2) generation of an “NO store” revealed by N-acetylcysteine-induced relaxation, and 3) formation of protein-bound dinitrosyl iron complexes in the medial layer of aortic rings exposed to LPS in vitro. In conclusion, the adventitia is a powerful source of NO triggered by LPS in the rat aorta. This novel source of NO has an important impact on smooth muscle function and might be implicated in various inflammatory diseases.

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A Second [2Fe-2S] Ferredoxin from Sphingomonas sp. Strain RW1 Can Function as an Electron Donor for the Dioxin Dioxygenase

Journal of Bacteriology ◽

10.1128/jb.182.8.2238-2244.2000 ◽

2000 ◽

Vol 182 (8) ◽

pp. 2238-2244 ◽

Cited By ~ 18

Author(s):

Jean Armengaud ◽

Jacques Gaillard ◽

Kenneth N. Timmis

Keyword(s):

Electron Donor ◽

Electron Paramagnetic Resonance Spectroscopy ◽

In Vitro Assays ◽

Resonance Spectroscopy ◽

Hydrogen Electrode ◽

Visible Absorption ◽

Paramagnetic Resonance ◽

Reading Frame

ABSTRACT The first step in the degradation of dibenzofuran and dibenzo-p-dioxin by Sphingomonas sp. strain RW1 is carried out by dioxin dioxygenase (DxnA1A2), a ring-dihydroxylating enzyme. An open reading frame (fdx3) that could potentially specify a new ferredoxin has been identified downstream ofdxnA1A2, a two-cistron gene (J. Armengaud, B. Happe, and K. N. Timmis, J. Bacteriol. 180:3954–3966, 1998). In the present study, we report a biochemical analysis of Fdx3 produced inEscherichia coli. This third ferredoxin thus far identified in Sphingomonas sp. strain RW1 contained a putidaredoxin-type [2Fe-2S] cluster which was characterized by UV-visible absorption spectrophotometry and electron paramagnetic resonance spectroscopy. The midpoint redox potential of this ferredoxin (E′0 = −247 ± 10 mV versus normal hydrogen electrode at pH 8.0) is similar to that exhibited by Fdx1 (−245 mV), a homologous ferredoxin previously characterized inSphingomonas sp. strain RW1. In in vitro assays, Fdx3 can be reduced by RedA2 (a reductase similar to class I cytochrome P-450 reductases), previously isolated from Sphingomonas sp. strain RW1. RedA2 exhibits a Km value of 3.2 ± 0.3 μM for Fdx3. In vivo coexpression of fdx3and redA2 with dxnA1A2 confirmed that Fdx3 can serve as an electron donor for the dioxin dioxygenase.

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In vitro Lipolysis as a Tool for the Establishment of IVIVC for Lipid-Based Drug Delivery Systems

Current Drug Delivery ◽

10.2174/1567201816666190620115716 ◽

2019 ◽

Vol 16 (8) ◽

pp. 688-697

Author(s):

Ravinder Verma ◽

Deepak Kaushik

Keyword(s):

Drug Delivery ◽

Ph Value ◽

Electron Paramagnetic Resonance Spectroscopy ◽

Rapid Screening ◽

Resonance Spectroscopy ◽

Fed State ◽

In Vitro Lipolysis ◽

Ph Stat

: In vitro lipolysis has emerged as a powerful tool in the development of in vitro in vivo correlation for Lipid-based Drug Delivery System (LbDDS). In vitro lipolysis possesses the ability to mimic the assimilation of LbDDS in the human biological system. The digestion medium for in vitro lipolysis commonly contains an aqueous buffer media, bile salts, phospholipids and sodium chloride. The concentrations of these compounds are defined by the physiological conditions prevailing in the fasted or fed state. The pH of the medium is monitored by a pH-sensitive electrode connected to a computercontrolled pH-stat device capable of maintaining a predefined pH value via titration with sodium hydroxide. Copenhagen, Monash and Jerusalem are used as different models for in vitro lipolysis studies. The most common approach used in evaluating the kinetics of lipolysis of emulsion-based encapsulation systems is the pH-stat titration technique. This is widely used in both the nutritional and the pharmacological research fields as a rapid screening tool. Analytical tools for the assessment of in vitro lipolysis include HPLC, GC, HPTLC, SEM, Cryo TEM, Electron paramagnetic resonance spectroscopy, Raman spectroscopy and Nanoparticle Tracking Analysis (NTA) for the characterization of the lipids and colloidal phases after digestion of lipids. Various researches have been carried out for the establishment of IVIVC by using in vitro lipolysis models. The current publication also presents an updated review of various researches in the field of in vitro lipolysis.

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