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Evaluation of putative nitrite anhydrase activity of carbonic anhydrase
Abstracts/Nitric Oxide 42 (2014) 99–153
CPTIO completely abrogates the activity of purified CA II in PRP, suggesting that the platelet inhibitory function of CA II is NO dependent (p = 0.04). In addition, inhibition of platelet activation by red blood cells and nitrite was significantly attenuated in the presence of dorzolamide (p < 0.05). These results demonstrate that erythrocytic CA II may play an important role in nitrite bioactivation by the red blood cell. However, Dorzolamide had no effect on NO production from RBCs and nitrite as measured by electron paramagnetic resonance and chemiluminescence, suggesting that Hb is the main producer of NO from nitrite in the red cell. Furthermore, we observed no significant difference in platelet inhibition by CA II under oxygenated vs deoxygenated conditions. This lack of an effect of oxygen tension is counter to the observed oxygen tension-dependence of nitritemediated platelet inhibition by red blood cells and isolated hemoglobin, where inhibition is favored under deoxygenated conditions. Thus, we propose that CA works in conjunction with Hb in the red cell to bioactivate nitrite. This work was supported by NIH Grants HL098032 and HL058091. Keywords: Mechanisms; Carbonic anhydrase; Nitrite; Nitric oxide; Hemoglobin; Platelets; NO; TRUSOPT; Dorzolamide; Plasma; RBC; Blood; CPTIO.
P205. Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide http://dx.doi.org/10.1016/j.niox.2014.09.146 Jun Wang a, Sabina Krizowski b, Katrin Fischer b, Dimitri Niks c, Jesus Tejero d, Courtney Sparacino-Watkins d, Ling Wang e, Venkata Ragireddy a, Sheila Frizzell a, Eric Kelley a, Yingze Zhang f, Partha Basu g, Russ Hille c, Guenter Schwarz b, Mark Gladwin f a University of Pittsburgh b Cologne University c University of California, Riverside d Heart, Lung, Blood and vascular Medicine Institute, University of Pittsburgh e Vascular Medicine Institute, University of Pittsburgh f University of Pittsburgh Medical Center g Duquesne University
Recent studies suggest that the molybdenum enzymes xanthine oxidase, aldehyde oxidase, mARC exhibit nitrite reductase activity at low partial oxygen pressures. However, inhibition studies of xanthine oxidase in humans have failed to block nitrite-dependent NO signaling, leading to continued exploration for other candidate nitrite reductases in humans. Another physiologically important molybdenum enzyme – sulfite oxidase (SO) – has not extensively been studied. Here we have performed studies to determine whether SO is an important nitrite reductase. Wild type and truncated forms of human SO were expressed in E. coli and purified with SourceQ15 columns. Analytic methods include chemiluminescent detection of NO/ nitrite, electron paramagnetic resonance on the molybdenum center, circular dichroism analysis of protein structure, western blot and cyclic guanosine monophosphate (cGMP) assays to analyze the role of SO in NO-sGC-cGMP pathway in human fibroblast. Effect of NO generation was evaluated in both a purging and non-purge chemiluminescent systems, showing similar quantitative results for NO production from either I3− -nitrite conversion or DETA-NONOate and better enzymatic steady-state kinetics in the non-purge system. Our results showed SO functioned as a nitrite reductase in the presence of a one-electron donor (phenosafranine), exhibiting redox-coupling of substrate oxidation and nitrite reduction to form NO (Km = 80 mM at pH 7.4). With sulfite, the physiological substrate, SO only facilitates one turnover of nitrite reduction. Electron paramagnetic resonance studies with heme and molybdenum domains indi-
149
cate that nitrite reduction occurs at the molybdenum center via coupled oxidation of Mo(IV) to Mo(V). Reaction rates of nitrite to NO decreased in the presence of a functional heme domain in SO by ~60% lower than the molybdenum domain alone, which was mediated by steric and redox effects of the heme domain. Using knock-down of all molybdopterin enzymes and SO in human fibroblasts, SO was found to contribute significantly to hypoxic nitrite signaling as demonstrated by ~80% decrease of cGMP in patient fibroblast compared with wild-type fibroblast. This work suggest that nitrite binds to and gets reduced at the molybdenum site of SO, an important molybdenum nitrite reductase, to form NO, and contributes to the mammalian nitrate-nitrite-NO signaling pathway. Keywords: Sulfite oxidase; Nitrite; Nitric oxide; Molybdenum; Heme; Sulfite; Phenosafranine; Hypoxia.
P206. Evaluation of putative nitrite anhydrase activity of carbonic anhydrase http://dx.doi.org/10.1016/j.niox.2014.09.147 Jun Wang a, Courtney Sparacino-Watkins b, Daniel Kim-Shapiro c, Mark Gladwin a a University of Pittsburgh b Heart, Lung, Blood and vascular Medicine Institute, University of Pittsburgh c Wake Forest University
The zinc-metalloenzyme carbonic anhydrase is the second most abundant protein in red blood cells. It exists in two isozymic forms, CAI and CAII. CAII is one of fastest enzymes, which catalyzes the reversible hydration of CO2 to generate bicarbonate anion (HCO3−) with maximal turnover number of 106 s−1 and Km of 10 mM for CO2. Recently CAII was reported to react with nitrite to form NO at low pH, which induced vasodilation in aortic rings. In this work, we explored the functional and kinetic behavior of CAII reactions with nitrite under different conditions. Chemiluminescent detection under argon (0% O2) or air (21% O2) was used for NO headspace detection. We observed that NO generation was nitrite dependant with low ν max (0.0067 nmol s−1 mg−1), Km (39 μM) and kcat (0.0002 s−1 at pH 7.2, 37 °C) in the presence of 10 μM CAII. Our studies showed that the rates of NO formation at pH 5.9 and 7.2 were not substantially different. When any classic CAII inhibitor was added to the reaction mixture, including sodium bicarbonate (0.5– 10 mM), dorzolamide (3 mM), acetazolamide (0.25 mM) or methazolamide (0.3 mM) at low mM concentrations, NO formation was incompletely inhibited, with an approximate 20– 50% decrease in NO formation in gas phase. The inhibitory effect of dorzolamide on CAII was also concentration dependent in the range of 0.25–3 mM. Furthermore, we observed similar NO production rates by CAII under oxygenated (21% O 2 ) and deoxygenated (0% O2) conditions. In addition, recombinant human CAII was expressed in E. coli, purified with FPLC, and evaluated for its nitrite anhydrase activity. Headspace NO analysis showed that bovine CAII had high activity than recombinant human CAII and CAI. Taken together, our data suggest CAII may generate NO from nitrite, but the reaction has a very low Vmax; contrary to prior publications suggesting that CA inhibitors enhance NO production, we see incomplete inhibition with CAII inhibitors. Keywords: Carbonic anhydrase; Nitrite; Nitric oxide; Dorzolamide; Bicarbonate; Normoxic; Hypoxic.
P207. NO-mediated interkingdom signaling in bacterial pathogenesis and controversies http://dx.doi.org/10.1016/j.niox.2014.09.148
CPTIO completely abrogates the activity of purified CA II in PRP, suggesting that the platelet inhibitory function of CA II is NO dependent (p = 0.04). In addition, inhibition of platelet activation by red blood cells and nitrite was significantly attenuated in the presence of dorzolamide (p < 0.05). These results demonstrate that erythrocytic CA II may play an important role in nitrite bioactivation by the red blood cell. However, Dorzolamide had no effect on NO production from RBCs and nitrite as measured by electron paramagnetic resonance and chemiluminescence, suggesting that Hb is the main producer of NO from nitrite in the red cell. Furthermore, we observed no significant difference in platelet inhibition by CA II under oxygenated vs deoxygenated conditions. This lack of an effect of oxygen tension is counter to the observed oxygen tension-dependence of nitritemediated platelet inhibition by red blood cells and isolated hemoglobin, where inhibition is favored under deoxygenated conditions. Thus, we propose that CA works in conjunction with Hb in the red cell to bioactivate nitrite. This work was supported by NIH Grants HL098032 and HL058091. Keywords: Mechanisms; Carbonic anhydrase; Nitrite; Nitric oxide; Hemoglobin; Platelets; NO; TRUSOPT; Dorzolamide; Plasma; RBC; Blood; CPTIO.
P205. Sulfite oxidase catalyzes single electron transfer at molybdenum domain to reduce nitrite to nitric oxide http://dx.doi.org/10.1016/j.niox.2014.09.146 Jun Wang a, Sabina Krizowski b, Katrin Fischer b, Dimitri Niks c, Jesus Tejero d, Courtney Sparacino-Watkins d, Ling Wang e, Venkata Ragireddy a, Sheila Frizzell a, Eric Kelley a, Yingze Zhang f, Partha Basu g, Russ Hille c, Guenter Schwarz b, Mark Gladwin f a University of Pittsburgh b Cologne University c University of California, Riverside d Heart, Lung, Blood and vascular Medicine Institute, University of Pittsburgh e Vascular Medicine Institute, University of Pittsburgh f University of Pittsburgh Medical Center g Duquesne University
Recent studies suggest that the molybdenum enzymes xanthine oxidase, aldehyde oxidase, mARC exhibit nitrite reductase activity at low partial oxygen pressures. However, inhibition studies of xanthine oxidase in humans have failed to block nitrite-dependent NO signaling, leading to continued exploration for other candidate nitrite reductases in humans. Another physiologically important molybdenum enzyme – sulfite oxidase (SO) – has not extensively been studied. Here we have performed studies to determine whether SO is an important nitrite reductase. Wild type and truncated forms of human SO were expressed in E. coli and purified with SourceQ15 columns. Analytic methods include chemiluminescent detection of NO/ nitrite, electron paramagnetic resonance on the molybdenum center, circular dichroism analysis of protein structure, western blot and cyclic guanosine monophosphate (cGMP) assays to analyze the role of SO in NO-sGC-cGMP pathway in human fibroblast. Effect of NO generation was evaluated in both a purging and non-purge chemiluminescent systems, showing similar quantitative results for NO production from either I3− -nitrite conversion or DETA-NONOate and better enzymatic steady-state kinetics in the non-purge system. Our results showed SO functioned as a nitrite reductase in the presence of a one-electron donor (phenosafranine), exhibiting redox-coupling of substrate oxidation and nitrite reduction to form NO (Km = 80 mM at pH 7.4). With sulfite, the physiological substrate, SO only facilitates one turnover of nitrite reduction. Electron paramagnetic resonance studies with heme and molybdenum domains indi-
149
cate that nitrite reduction occurs at the molybdenum center via coupled oxidation of Mo(IV) to Mo(V). Reaction rates of nitrite to NO decreased in the presence of a functional heme domain in SO by ~60% lower than the molybdenum domain alone, which was mediated by steric and redox effects of the heme domain. Using knock-down of all molybdopterin enzymes and SO in human fibroblasts, SO was found to contribute significantly to hypoxic nitrite signaling as demonstrated by ~80% decrease of cGMP in patient fibroblast compared with wild-type fibroblast. This work suggest that nitrite binds to and gets reduced at the molybdenum site of SO, an important molybdenum nitrite reductase, to form NO, and contributes to the mammalian nitrate-nitrite-NO signaling pathway. Keywords: Sulfite oxidase; Nitrite; Nitric oxide; Molybdenum; Heme; Sulfite; Phenosafranine; Hypoxia.
P206. Evaluation of putative nitrite anhydrase activity of carbonic anhydrase http://dx.doi.org/10.1016/j.niox.2014.09.147 Jun Wang a, Courtney Sparacino-Watkins b, Daniel Kim-Shapiro c, Mark Gladwin a a University of Pittsburgh b Heart, Lung, Blood and vascular Medicine Institute, University of Pittsburgh c Wake Forest University
The zinc-metalloenzyme carbonic anhydrase is the second most abundant protein in red blood cells. It exists in two isozymic forms, CAI and CAII. CAII is one of fastest enzymes, which catalyzes the reversible hydration of CO2 to generate bicarbonate anion (HCO3−) with maximal turnover number of 106 s−1 and Km of 10 mM for CO2. Recently CAII was reported to react with nitrite to form NO at low pH, which induced vasodilation in aortic rings. In this work, we explored the functional and kinetic behavior of CAII reactions with nitrite under different conditions. Chemiluminescent detection under argon (0% O2) or air (21% O2) was used for NO headspace detection. We observed that NO generation was nitrite dependant with low ν max (0.0067 nmol s−1 mg−1), Km (39 μM) and kcat (0.0002 s−1 at pH 7.2, 37 °C) in the presence of 10 μM CAII. Our studies showed that the rates of NO formation at pH 5.9 and 7.2 were not substantially different. When any classic CAII inhibitor was added to the reaction mixture, including sodium bicarbonate (0.5– 10 mM), dorzolamide (3 mM), acetazolamide (0.25 mM) or methazolamide (0.3 mM) at low mM concentrations, NO formation was incompletely inhibited, with an approximate 20– 50% decrease in NO formation in gas phase. The inhibitory effect of dorzolamide on CAII was also concentration dependent in the range of 0.25–3 mM. Furthermore, we observed similar NO production rates by CAII under oxygenated (21% O 2 ) and deoxygenated (0% O2) conditions. In addition, recombinant human CAII was expressed in E. coli, purified with FPLC, and evaluated for its nitrite anhydrase activity. Headspace NO analysis showed that bovine CAII had high activity than recombinant human CAII and CAI. Taken together, our data suggest CAII may generate NO from nitrite, but the reaction has a very low Vmax; contrary to prior publications suggesting that CA inhibitors enhance NO production, we see incomplete inhibition with CAII inhibitors. Keywords: Carbonic anhydrase; Nitrite; Nitric oxide; Dorzolamide; Bicarbonate; Normoxic; Hypoxic.
P207. NO-mediated interkingdom signaling in bacterial pathogenesis and controversies http://dx.doi.org/10.1016/j.niox.2014.09.148