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Intramolecular electron transfer controls nitrite reduction in molybdenum-containing sulfite oxidase
Abstracts/Nitric Oxide 42 (2014) 99–153
The chaperone heat shock protein 90 (hsp90) associates with signaling proteins in cells including soluble guanylate cyclase (sGC). Hsp90 associates with the heme-free (apo) sGC-β1 subunit and helps to drive heme insertion as required for maturation of sGC to its nitric oxide (NO)-responsive active form. Here we found that NO caused apo-sGC-β1 to rapidly and transiently dissociate from hsp90 and associate with sGC-α1 in cells. This NO response: (i) Required that hsp90 be active and that cellular heme be available and be capable of inserting into apo-sGCβ1; (ii) was associated with an increase in sGC-β1 heme content; (iii) could be mimicked by the heme-independent sGC activator BAY 60–2770; (iv) was followed by desensitization of sGC toward NO, sGC-α1 disassociation, and reassociation with hsp90. Thus, NO promoted a rapid, transient, and hsp90dependent heme insertion into the apo-sGC-β1 subpopulation in cells, which enabled it to combine with the sGC-α1 subunit to form the mature enzyme. The driving mechanism likely involves conformational changes near the heme site in sGC-β1 that can be mimicked by the pharmacologic sGC activator. Such dynamic interplay between hsp90, apo-sGC-β1, and sGCα1 in response to NO is unprecedented, and represent new steps by which cells can modulate heme content and activity of sGC for signaling cascades in physiologic or pathologic settings. Keywords: Nitric oxide; Heme; Hsp90; Guanylate cyclase; Desensitization.
Oral 2021-1. Intramolecular electron transfer controls nitrite reduction in molybdenum-containing sulfite oxidase http://dx.doi.org/10.1016/j.niox.2014.09.045 Guenter Schwarz a, Sabina Krizowski a, Jun Wang b, Dimitri Niks c, Courtney Sparacino-Watkins d, Russ Hille c, Mark Gladwin b a Cologne University b University of Pittsburgh c University of California at Riverside d Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh
Nitric oxide (NO) is a unique second messenger that controls fundamental biological functions such as blood pressure, hypoxic vasodilation and mitochondrial respiration. Besides the classical oxygen-dependent arginine NO-synthase pathway, NO can be generated from nitrite, representing an important alternative source of NO under oxygen limited conditions. Recently we have identified mitochondrial sulfite oxidase (SO), one of four molybdenum-dependent enzymes as a new nitrite reductase that is able to generate NO under physiological conditions (see abstract of Jun et al.). Recombinant sulfite-reduced human SO reduced nitrite at the molybdenum domain with a one-electron oxidation of MoIV to MoV. The heme domain of SO was shown to impair nitrite-reducing activity, which was based on electronic hindrance because nitrite and the heme iron compete for the same Mo-derived electron. Consequently, the idea that a restricted intra-molecular electron transfer (IET) from Mo to heme would increase the NO synthesis rate of SO, was investigated by two different approaches. The first is based on truncations of the inter-domain tether that controls conformational flexibility, which is required for fast IET. Alternatively, surface residues were identified in the interface of the Mo and heme domain binding site that were shown to be directly involved in IET between both metal centers. SO variants that were either altered in the tether or the domain binding site were generated and characterized regarding their kinetic parameters and nitritedependent NO synthesis. Both IET restricted approaches support the view that with decreasing IET, nitrite-dependent NO syn-
113
thesis is increased, thus representing a novel target for drug development. Furthermore, we demonstrate that SO significantly contributes to nitrite-dependent NO signaling in fibroblast, by measuring NO-dependent cGMP levels, produced via the NOsoluble guanylyl cyclase pathway. Finally, the expression of SO in human endothelial cells was identified by quantitative PCR and Western blot, suggesting that SO contributes to the mammalian nitrite–NO pathway in blood vessels, the regulatory side of vascular tone. In conclusion, we provide in vitro and in vivo evidence for a mechanism that controls nitrite reduction by SO in vascular and epithelial tissue. Keywords: Sulfite oxidase; Nitrite; Nitric oxide; Molybdenum cofactor; Heme; Sulfite; Intramolecular electron transfer; Domain movement; Hypoxia.
Oral 2021-2. Role of nitrite and sulfide metabolites in the cardiovascular system of aged mice http://dx.doi.org/10.1016/j.niox.2014.09.046 Gopi Kolluru, Saurabh Rajpal, Sibile Pardue, Christopher Kevil LSU Health Sciences Center
Objective/purpose: Aging is associated with various complications including oxidative stress and inflammation. It is considered a major risk factor for neurodegenerative and cardiovascular diseases (CVD). Hydrogen sulfide (H2S) and nitric oxide (NO) are gaseous signaling molecules implicated in several pathophysiological functions of the vasculature. Reduced bioavailability of NO and shortening of telomere length with age is well known. Increase SIRT1 is known to slow the process of aging. H2S is shown to inhibit free radicals and regulate SIRT1. We sought to determine the age dependent regulation of H2S and NO metabolite bioavailability in mice and rescue mechanisms mediated upon H 2 S therapy in aged mice. Methods: H 2 S and NO metabolites were measured in various organs of C57BL/6J wild type (WT) mice at age 6, 12, 24 and 52 weeks. Unilateral femoral artery ligation was performed on 52-week-old WT mice, which were treated with PBS or H2S donor DATS (200 mg/kg) administered retro-orbitally twice daily until Day 7. Hind limb perfusion was measured by laser Doppler perfusion flowmetry. Tissue angiogenic index and mature vessel density was determined by the ratios of CD31/ DAPI and α-SMA/CD31 staining. The monobromobimane (MBB)HPLC method was used to measure various sulfide pools – free, acid labile sulfides and bound sulfane sulfur pools. NO metabolites, nitrite and X-NO, were measured using NO chemiluminescence. Results: In the heart, aorta, carotid artery, mesenteric artery there was a significant decrease in total sulfide levels with age over 52 weeks. This decrease in sulfide levels was not particularly observed in kidney and lungs. NO metabolites also decreased with age in the aorta, mesenteric artery, carotid artery and heart. This reduction was more prominent in the X-NO pool. Perfusion of ischemic hind limb was increased by DATS. The angiogenic index and mature vessel density was significantly increased in aged WT mice after treatment with DATS. Conclusion: Our study clearly reveals that both H2S and NO metabolites decrease with aging in the cardiovascular system. There is a differential effect of aging on various biochemical forms of H2S and NO. H2S may play an important role in rescue mechanisms including ischemic revascularization of age-related effects. Keywords: Nitrite; Sulfide; Aging; Cardiovascular system; Ischemia; Vascular remodeling.
The chaperone heat shock protein 90 (hsp90) associates with signaling proteins in cells including soluble guanylate cyclase (sGC). Hsp90 associates with the heme-free (apo) sGC-β1 subunit and helps to drive heme insertion as required for maturation of sGC to its nitric oxide (NO)-responsive active form. Here we found that NO caused apo-sGC-β1 to rapidly and transiently dissociate from hsp90 and associate with sGC-α1 in cells. This NO response: (i) Required that hsp90 be active and that cellular heme be available and be capable of inserting into apo-sGCβ1; (ii) was associated with an increase in sGC-β1 heme content; (iii) could be mimicked by the heme-independent sGC activator BAY 60–2770; (iv) was followed by desensitization of sGC toward NO, sGC-α1 disassociation, and reassociation with hsp90. Thus, NO promoted a rapid, transient, and hsp90dependent heme insertion into the apo-sGC-β1 subpopulation in cells, which enabled it to combine with the sGC-α1 subunit to form the mature enzyme. The driving mechanism likely involves conformational changes near the heme site in sGC-β1 that can be mimicked by the pharmacologic sGC activator. Such dynamic interplay between hsp90, apo-sGC-β1, and sGCα1 in response to NO is unprecedented, and represent new steps by which cells can modulate heme content and activity of sGC for signaling cascades in physiologic or pathologic settings. Keywords: Nitric oxide; Heme; Hsp90; Guanylate cyclase; Desensitization.
Oral 2021-1. Intramolecular electron transfer controls nitrite reduction in molybdenum-containing sulfite oxidase http://dx.doi.org/10.1016/j.niox.2014.09.045 Guenter Schwarz a, Sabina Krizowski a, Jun Wang b, Dimitri Niks c, Courtney Sparacino-Watkins d, Russ Hille c, Mark Gladwin b a Cologne University b University of Pittsburgh c University of California at Riverside d Heart, Lung, Blood and Vascular Medicine Institute, University of Pittsburgh
Nitric oxide (NO) is a unique second messenger that controls fundamental biological functions such as blood pressure, hypoxic vasodilation and mitochondrial respiration. Besides the classical oxygen-dependent arginine NO-synthase pathway, NO can be generated from nitrite, representing an important alternative source of NO under oxygen limited conditions. Recently we have identified mitochondrial sulfite oxidase (SO), one of four molybdenum-dependent enzymes as a new nitrite reductase that is able to generate NO under physiological conditions (see abstract of Jun et al.). Recombinant sulfite-reduced human SO reduced nitrite at the molybdenum domain with a one-electron oxidation of MoIV to MoV. The heme domain of SO was shown to impair nitrite-reducing activity, which was based on electronic hindrance because nitrite and the heme iron compete for the same Mo-derived electron. Consequently, the idea that a restricted intra-molecular electron transfer (IET) from Mo to heme would increase the NO synthesis rate of SO, was investigated by two different approaches. The first is based on truncations of the inter-domain tether that controls conformational flexibility, which is required for fast IET. Alternatively, surface residues were identified in the interface of the Mo and heme domain binding site that were shown to be directly involved in IET between both metal centers. SO variants that were either altered in the tether or the domain binding site were generated and characterized regarding their kinetic parameters and nitritedependent NO synthesis. Both IET restricted approaches support the view that with decreasing IET, nitrite-dependent NO syn-
113
thesis is increased, thus representing a novel target for drug development. Furthermore, we demonstrate that SO significantly contributes to nitrite-dependent NO signaling in fibroblast, by measuring NO-dependent cGMP levels, produced via the NOsoluble guanylyl cyclase pathway. Finally, the expression of SO in human endothelial cells was identified by quantitative PCR and Western blot, suggesting that SO contributes to the mammalian nitrite–NO pathway in blood vessels, the regulatory side of vascular tone. In conclusion, we provide in vitro and in vivo evidence for a mechanism that controls nitrite reduction by SO in vascular and epithelial tissue. Keywords: Sulfite oxidase; Nitrite; Nitric oxide; Molybdenum cofactor; Heme; Sulfite; Intramolecular electron transfer; Domain movement; Hypoxia.
Oral 2021-2. Role of nitrite and sulfide metabolites in the cardiovascular system of aged mice http://dx.doi.org/10.1016/j.niox.2014.09.046 Gopi Kolluru, Saurabh Rajpal, Sibile Pardue, Christopher Kevil LSU Health Sciences Center
Objective/purpose: Aging is associated with various complications including oxidative stress and inflammation. It is considered a major risk factor for neurodegenerative and cardiovascular diseases (CVD). Hydrogen sulfide (H2S) and nitric oxide (NO) are gaseous signaling molecules implicated in several pathophysiological functions of the vasculature. Reduced bioavailability of NO and shortening of telomere length with age is well known. Increase SIRT1 is known to slow the process of aging. H2S is shown to inhibit free radicals and regulate SIRT1. We sought to determine the age dependent regulation of H2S and NO metabolite bioavailability in mice and rescue mechanisms mediated upon H 2 S therapy in aged mice. Methods: H 2 S and NO metabolites were measured in various organs of C57BL/6J wild type (WT) mice at age 6, 12, 24 and 52 weeks. Unilateral femoral artery ligation was performed on 52-week-old WT mice, which were treated with PBS or H2S donor DATS (200 mg/kg) administered retro-orbitally twice daily until Day 7. Hind limb perfusion was measured by laser Doppler perfusion flowmetry. Tissue angiogenic index and mature vessel density was determined by the ratios of CD31/ DAPI and α-SMA/CD31 staining. The monobromobimane (MBB)HPLC method was used to measure various sulfide pools – free, acid labile sulfides and bound sulfane sulfur pools. NO metabolites, nitrite and X-NO, were measured using NO chemiluminescence. Results: In the heart, aorta, carotid artery, mesenteric artery there was a significant decrease in total sulfide levels with age over 52 weeks. This decrease in sulfide levels was not particularly observed in kidney and lungs. NO metabolites also decreased with age in the aorta, mesenteric artery, carotid artery and heart. This reduction was more prominent in the X-NO pool. Perfusion of ischemic hind limb was increased by DATS. The angiogenic index and mature vessel density was significantly increased in aged WT mice after treatment with DATS. Conclusion: Our study clearly reveals that both H2S and NO metabolites decrease with aging in the cardiovascular system. There is a differential effect of aging on various biochemical forms of H2S and NO. H2S may play an important role in rescue mechanisms including ischemic revascularization of age-related effects. Keywords: Nitrite; Sulfide; Aging; Cardiovascular system; Ischemia; Vascular remodeling.