Assay and Characterization of the NO Dioxygenase Activity of Flavohemoglobins

doi:10.1016/S0076-6879(08)36012-1

Methods in Enzymology

Volume 436, 2008, Pages 217-237

https://doi.org/10.1016/S0076-6879(08)36012-1 Get rights and content

Abstract

A variety of hemoglobins, including several microbial flavohemoglobins, enzymatically dioxygenate the free radical nitric oxide (•NO) to form nitrate. Many of these •NO dioxygenases have been shown to control •NO toxicity and signaling. Furthermore, •NO dioxygenation appears to be an ancient and intrinsic function for members of the hemoglobin superfamily found in Archaea, eukaryotes, and bacteria. Yet for many hemoglobins, a function remains to be elucidated. Methods for the assay and characterization of the •NO dioxygenase (EC 1.14.12.17) activity and function of flavohemoglobins are described. The methods may also be applied to the discovery and design of inhibitors for use as antibiotics or as modulators of •NO signaling.

Section snippets

INTRODUCTION

Evidence suggests that the earliest hemoglobin functioned not as an O₂ transport or storage protein, but as an enzyme with the capacity to dioxygenate and detoxify •NO (Gardner 2005, Gardner et al., 1998b, Wu et al., 2003). Moreover, this primitive enzymatic activity and function appears to have been retained in the modern O₂ transport or storage hemoglobin and myoglobin during approximately 2 billion years of evolution (Miranda et al., 2005, Vinogradov et al., 2006). Thus, several of the

THE •NO DIOXYGENASE MECHANISM

The proposed dioxygenase mechanism for the enzymatic conversion of •NO to nitrate by hemoglobins and myoglobins was recently reviewed and will not be extensively discussed here (Gardner 2005, Gardner et al., 2006, Olson et al., 2004). Here, I focus on the methods used for characterization of a •NO dioxygenase activity as well as the issues and difficulties one can expect to encounter when trying to assign a •NO dioxygenase function to various flavohemoglobins. Nevertheless, it is important to

SUSCEPTIBILITY OF THE •NO DIOXYGENASE TO •NO INHIBITION

In order for a hemoglobin to function as an efficient •NO dioxygenase, it must avoid or limit the inhibition caused by the high affinity binding of •NO to the ferrous heme [Eq. (12.6)] in place of the substrate O₂ [Eq. (12.2)] (Gardner et al., 2000a, Gardner et al., 2000b). Flavohemoglobins can avoid, but not totally escape, •NO inhibition by binding O₂ with high affinity or by reducing •NO to nitroxyl (NO⁻) [Eq. (12.7)].

The importance of a high O₂ affinity is best illustrated by the effect of

AUTOOXIDATION OF HEMOGLOBINS AND •NO DECOMPOSITION

In addition, mechanisms for limiting autooxidation and ⁻O₂• generation by hemoglobin [Eq. (12.9)] are critical for an •NO dioxygenase function. High rates of ⁻O₂• release necessarily increase cellular ⁻O₂• levels, which is counterproductive for organisms in the absence of sufficient superoxide dismutase to scavenge the excess toxic ⁻O₂• (Fridovich, 1995). •NO reacts with free ⁻O₂• with a second‐order rate constant of 6.7 × 10⁹ M⁻¹s⁻¹ to form highly reactive and toxic peroxynitrite [Eq. (12.8)] (

METHEMOGLOBIN REDUCTION

Perhaps no issue demands greater attention than the cellular reducing systems for the various single‐domain hemoglobins and myoglobins acting as dioxygenases [Eq. (12.5)]. In the erythrocyte, hemoglobin is reduced by a FAD‐containing methemoglobin reductase and cytochrome b₅ (Hultquist and Passon, 1971), but the reducing system for myoglobin acting as a •NO dioxygenase within myocytes remains poorly defined (Flögel et al., 2004, Flögel et al., 2001). Cytochrome b₅ can reduce metmyoglobin and is

HEME AND FLAVIN COFACTORS

It has become increasingly evident that the content of the heme and FAD cofactors in isolated flavohemoglobins, and heme in isolated hemoglobins, is more often than not substoichiometric. Although initially unexpected and troubling for the work on flavohemoglobins (Gardner et al., 1998b), there is now ample evidence that heme and FAD can be substoichiometric within cells or in isolated flavohemoglobins (Kobayashi et al., 2002). For E. coli flavohemoglobin, one can observe the separation of the

Reagents

100 mM potassium phosphate buffer, pH 7.0, containing 0.3 mM EDTA
•NO (2 mM) prepared in water and stored at 4° (see subsequent section)
O₂ (1.14 mM) prepared in phosphate buffer (see subsequent section)
CO (1 mM) prepared in water and stored at 4° (see subsequent section)
NADH or NADPH (10 mM) prepared fresh in water
Manganese‐containing superoxide dismutase (E. coli) (100 mg/ml) in potassium phosphate buffer and stored at −80°
FAD (1 mM) prepared in water and stored at −20°
1 M glucose in water stored at −20°

Reagents and materials

98.5% •NO gas lecture bottle
99.9% O₂ gas tank
99.5% CO gas lecture bottle
99.99% Ar or ultrapure N₂ gas tank
AG 1‐X8 anion exchange resin (200 to 400 mesh acetate form) stored at 4°
Potassium permanganate (0.16 M) and 2.5% sulfuric acid prepared freshly in water
Potassium phosphate (100 mM) buffer, pH 7.0, containing 0.3 mM EDTA
Stainless‐steel gas proportioner/flow regulator
Vacutainer tubes (10 mm × 100 mm)

Rubber septum‐sealed glass tubes containing 3 mm × 10 mm micro stir bars and 1 ml pure water are used

Reagents

FAD standard (1.0 mM) prepared in water and stored at −20°
Pyridine (4.4 M) prepared in NaOH (0.2 M) and stored at room temperature
Dithionite (solid)
Potassium ferricyanide (100 mM) prepared in water and stored at −20°

The FAD content of flavohemoglobin is determined by first boiling the flavohemoglobin (10 to 20 nmol protein) in 1 mL of 100 mM potassium phosphate, pH 7.0, buffer containing 0.3 mM EDTA for 3 min. The denatured protein is then removed by centrifugation at 20,000 ×g for 3 min at room

Reagents

20 mM hemin prepared in dimethyl sulfoxide and stored at −20°
100 mM dithiothreitol in water stored at −20°
Dithionite (solid)
Tris‐Cl (50 mM), pH 8, containing EDTA (1 mM)
Catalase (bovine liver) (2,60,000 units per mL)

To reconstitute heme‐deficient flavohemoglobin with heme, flavohemoglobin is incubated with heme under reducing conditions in the presence of catalase to scavenge potentially damaging peroxide. Briefly, flavohemoglobin (0.5 to 0.75 mM) is prepared in a volume of 1.5 mL containing 50 mM

Reagents

Tris‐Cl, pH 7.5 (50 mM)
NADPH (solid)
Nitrate reductase (Aspergillus niger) (30 mg/mL in Tris‐Cl buffer) stored at −80°
Sodium nitrite standard (10 mM) prepared in water and stored at 4°
Sodium nitrate standard (10 mM) prepared in water and stored at 4°
Sulfanilamide (1% w/v) in phosphoric acid (5% v/v) and stored for weeks at 4°
N‐(1‐naphthyl‐) ethylenediamine 2HCl (0.1% w/v) in water stored for weeks at 4°

•NO‐saturated water prepared over AG 1‐X8 anion exchange resin is used to measure nitrate and

CONCLUSIONS

Hemoglobins and myoglobins bear an intrinsic capacity for high fidelity •NO dioxygenation. Moreover, •NO dioxygenation by hemoglobins may serve important roles in modulating •NO signaling and in limiting •NO toxicity. While evidence suggests that many flavohemoglobins from diverse sources function as •NO dioxygenases, many hemoglobins, including neuroglobin and cytoglobin, remain to be extensively tested for this function both in vitro and within cells. Reducing systems for single‐domain

ACKNOWLEDGMENTS

I gratefully acknowledge the past support of Grant GM65090 from the National Institutes of Health. I especially thank my coworkers, many of whom are listed in the cited publications, for their valuable contributions.

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Dioxygen and glucose force motion of the electron-transfer switch in the iron(III) flavohemoglobin-type nitric oxide dioxygenase
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Kinetic and structural investigations of the flavohemoglobin-type NO dioxygenase have suggested critical roles for transient Fe(III)O₂ complex formation and O₂-forced movements affecting hydride transfer to the FAD cofactor and electron-transfer to the Fe(III)O₂ complex. Stark-effect theory together with structural models and dipole and internal electrostatic field determinations provided a semi-quantitative spectroscopic method for investigating the proposed Fe(III)O₂ complex and O₂-forced movements. Deoxygenation of the enzyme causes Stark effects on the ferric heme Soret and charge-transfer bands revealing the Fe(III)O₂ complex. Deoxygenation also elicits Stark effects on the FAD that expose forces and motions that create a more restricted NADH access to FAD for hydride transfer and switch electron-transfer off. Glucose also forces the enzyme toward an off state. Amino acid substitutions at the B10, E7, E11, G8, D5, and F7 positions influence the Stark effects of O₂ on resting heme spin states and FAD consistent with the proposed roles of the side chains in the enzyme mechanism. Deoxygenation of ferric myoglobin and hemoglobin A also induces Stark effects on the hemes suggesting a common ‘oxy-met’ state. The ferric myoglobin and hemoglobin heme spectra are also glucose-responsive. A conserved glucose or glucose-6-phosphate binding site is found bridging the BC-corner and G-helix in flavohemoglobin and myoglobin suggesting novel allosteric effector roles for glucose or glucose-6-phosphate in the NO dioxygenase and O₂ storage functions. The results support the proposed roles of a ferric O₂ intermediate and protein motions in regulating electron-transfer during NO dioxygenase turnover.
Allostery in the nitric oxide dioxygenase mechanism of flavohemoglobin
2021, Journal of Biological Chemistry
The substrates O₂ and NO cooperatively activate the NO dioxygenase function of Escherichia coli flavohemoglobin. Steady-state and transient kinetic measurements support a structure-based mechanistic model in which O₂ and NO movements and conserved amino acids at the E11, G8, E2, E7, B10, and F7 positions within the globin domain control activation. In the cooperative and allosteric mechanism, O₂ migrates to the catalytic heme site via a long hydrophobic tunnel and displaces LeuE11 away from the ferric iron, which forces open a short tunnel to the catalytic site gated by the ValG8/IleE15 pair and LeuE11. NO permeates this tunnel and leverages upon the gating side chains triggering the CD loop to furl, which moves the E and F-helices and switches an electron transfer gate formed by LysF7, GlnE7, and water. This allows FADH₂ to reduce the ferric iron, which forms the stable ferric–superoxide–TyrB10/GlnE7 complex. This complex reacts with internalized NO with a bimolecular rate constant of 10¹⁰ M⁻¹ s⁻¹ forming nitrate, which migrates to the CD loop and unfurls the spring-like structure. To restart the cycle, LeuE11 toggles back to the ferric iron. Actuating electron transfer with O₂ and NO movements averts irreversible NO poisoning and reductive inactivation of the enzyme. Together, structure snapshots and kinetic constants provide glimpses of intermediate conformational states, time scales for motion, and associated energies.
Increasing the endogenous NO level causes catalase inactivation and reactivation of intercellular apoptosis signaling specifically in tumor cells
2015, Redox Biology
Tumor cells generate extracellular superoxide anions and are protected against intercellular apoptosis-inducing HOCl- and NO/peroxynitrite signaling through the expression of membrane-associated catalase. This enzyme decomposes H₂O₂ and thus prevents HOCl synthesis. It efficiently interferes with NO/peroxynitrite signaling through oxidation of NO and decomposition of peroxynitrite. The regulatory potential of catalase at the crosspoint of ROS and RNS chemical biology, as well as its high local concentration on the outside of the cell membrane of tumor cells, establish tight control of intercellular signaling and thus prevent tumor cell apoptosis. Therefore, inhibition of catalase or its inactivation by singlet oxygen reactivate intercellular apoptosis-inducing signaling. Nitric oxide and peroxynitrite are connected with catalase in multiple and meaningful ways, as (i) NO can be oxidated by compound I of catalase, (ii) NO can reversibly inhibit catalase, (iii) peroxynitrite can be decomposed by catalase and (iv) the interaction between peroxynitrite and H₂O₂ leads to the generation of singlet oxygen that inactivates catalase. Therefore, modulation of the concentration of free NO through addition of arginine, inhibition of arginase, induction of NOS expression or inhibition of NO dioxygenase triggers an autoamplificatory biochemical cascade that is based on initial formation of singlet oxygen, amplification of superoxide anion/H₂O₂ and NO generation through singlet oxygen dependent stimulation of the FAS receptor and caspase-8. Finally, singlet oxygen is generated at sufficiently high concentration to inactivate protective catalase and to reactivate intercellular apoptosis-inducing ROS signaling. This regulatory network allows to establish several pathways for synergistic interactions, like the combination of modulators of NO metabolism with enhancers of superoxide anion generation, modulators of NO metabolism that act at different targets and between modulators of NO metabolism and direct catalase inhibitors. The latter aspect is explicitely studied for the interaction between catalase inhibiting acetylsalicylic acid and an NO donor. It is also shown that hybrid molecules like NO-aspirin utilize this synergistic potential. Our data open novel approaches for rational tumor therapy based on specific ROS signaling and its control in tumor cells.
Globins scavenge sulfur trioxide anion radical
2015, Journal of Biological Chemistry
Citation Excerpt :
Catalase was resuspended in acetate buffer (pH 5.8), concentrated, filter-sterilized, and stored at 4 °C. CO-saturated water stocks were prepared as described (26). Superdex 200 was obtained from GE Healthcare Life Sciences.
Ferrous myoglobin was oxidized by sulfur trioxide anion radical (STAR) during the free radical chain oxidation of sulfite. Oxidation was inhibited by the STAR scavenger GSH and by the heme ligand CO. Bimolecular rate constants for the reaction of STAR with several ferrous globins and biomolecules were determined by kinetic competition. Reaction rate constants for myoglobin, hemoglobin, neuroglobin, and flavohemoglobin are large at 38, 120, 2,600, and ≥ 7,500 × 10⁶ m⁻¹ s⁻¹, respectively, and correlate with redox potentials. Measured rate constants for O₂, GSH, ascorbate, and NAD(P)H are also large at ∼100, 10, 130, and 30 × 10⁶ m⁻¹ s⁻¹, respectively, but nevertheless allow for favorable competition by globins and a capacity for STAR scavenging in vivo. Saccharomyces cerevisiae lacking sulfite oxidase and deleted of flavohemoglobin showed an O₂-dependent growth impairment with nonfermentable substrates that was exacerbated by sulfide, a precursor to mitochondrial sulfite formation. Higher O₂ exposures inactivated the superoxide-sensitive mitochondrial aconitase in cells, and hypoxia elicited both aconitase and NADP⁺-isocitrate dehydrogenase activity losses. Roles for STAR-derived peroxysulfate radical, superoxide radical, and sulfo-NAD(P) in the mechanism of STAR toxicity and flavohemoglobin protection in yeast are suggested.
Flavohemoglobin and nitric oxide detoxification in the human protozoan parasite Giardia intestinalis
2010, Biochemical and Biophysical Research Communications
Citation Excerpt :
Afterwards, 1 × 106 cells or 0.1 nM flavoHb were added and the NO consumption monitored. According to [31], activity was calculated at [NO] = 1 μM and corrected for the background rate of NO consumption by O2 as independently measured at the same NO concentration in the absence of cells or flavoHb. Whole-cell extracts were prepared from Giardia cells and, typically, the equivalent of 1 × 106 cells was loaded per lane.
Flavohemoglobins (flavoHbs), commonly found in bacteria and fungi, afford protection from nitrosative stress by degrading nitric oxide (NO) to nitrate. Giardia intestinalis, a microaerophilic parasite causing one of the most common intestinal human infectious diseases worldwide, is the only pathogenic protozoon as yet identified coding for a flavoHb. By NO amperometry we show that, in the presence of NADH, the recombinant Giardia flavoHb metabolizes NO with high efficacy under aerobic conditions (TN = 116 ± 10 s⁻¹ at 1 μM NO, T = 37 °C). The activity is [O₂]-dependent and characterized by an apparent K_M,O2 = 22 ± 7 μM. Immunoblotting analysis shows that the protein is expressed at low levels in the vegetative trophozoites of Giardia; accordingly, these cells aerobically metabolize NO with low efficacy. Interestingly, in response to nitrosative stress (24-h incubation with ⩾5 mM nitrite) flavoHb expression is enhanced and the trophozoites thereby become able to metabolize NO efficiently, the activity being sensitive to both cyanide and carbon monoxide. The NO-donors S-nitrosoglutathione (GSNO) and DETA-NONOate mimicked the effect of nitrite on flavoHb expression. We propose that physiologically flavoHb contributes to NO detoxification in G. intestinalis.
Nitric-oxide dioxygenase function of human cytoglobin with cellular reductants and in rat hepatocytes
2010, Journal of Biological Chemistry
Citation Excerpt :
NO-saturated water (1.94 mm) was prepared over AG 1-X8 resin as previously described (34). CO-saturated water (1.0 mm) was prepared under 99.5% CO (34). O2-saturated buffer (1.14 mm) was prepared, and O2 concentrations were varied in reactions as previously described (27, 34, 36).
Cytoglobin (Cygb) was investigated for its capacity to function as a NO dioxygenase (NOD) in vitro and in hepatocytes. Ascorbate and cytochrome b₅ were found to support a high NOD activity. Cygb-NOD activity shows respective K_m values for ascorbate, cytochrome b₅, NO, and O₂ of 0.25 mm, 0.3 μm, 40 nm, and ∼20 μm and achieves a k_cat of 0.5 s⁻¹. Ascorbate and cytochrome b₅ reduce the oxidized Cygb-NOD intermediate with apparent second order rate constants of 1000 m⁻¹ s⁻¹ and 3 × 10⁶ m⁻¹ s⁻¹, respectively. In rat hepatocytes engineered to express human Cygb, Cygb-NOD activity shows a similar k_cat of 1.2 s⁻¹, a K_m(NO) of 40 nm, and a k_cat/K_m(NO) (k′_NOD) value of 3 × 10⁷ m⁻¹ s⁻¹, demonstrating the efficiency of catalysis. NO inhibits the activity at [NO]/[O₂] ratios >1:500 and limits catalytic turnover. The activity is competitively inhibited by CO, is slowly inactivated by cyanide, and is distinct from the microsomal NOD activity. Cygb-NOD provides protection to the NO-sensitive aconitase. The results define the NOD function of Cygb and demonstrate roles for ascorbate and cytochrome b₅ as reductants.

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Chapter Twelve - Assay and Characterization of the NO Dioxygenase Activity of Flavohemoglobins

Abstract

Section snippets

INTRODUCTION

THE •NO DIOXYGENASE MECHANISM

SUSCEPTIBILITY OF THE •NO DIOXYGENASE TO •NO INHIBITION

AUTOOXIDATION OF HEMOGLOBINS AND •NO DECOMPOSITION

METHEMOGLOBIN REDUCTION

HEME AND FLAVIN COFACTORS

Reagents

Reagents and materials

Reagents

Reagents

Reagents

CONCLUSIONS

ACKNOWLEDGMENTS

Meth. Enzymol.

FASEB J.

J. Biol. Chem.

J. Biol. Chem.

Meth. Enzymol.

J. Inorg. Biochem.

J. Biol. Chem.

J. Biol. Chem.

Proc. Natl. Acad. Sci. USA

J. Biol. Chem.

Anal. Biochem.

Free Radic. Biol. Med.

Proc. Natl. Acad. Sci. USA

Free Radic. Res. Commun.

J. Biol. Inorg. Chem.

J. Biol. Chem.

Phytochemistry

Free Radic. Biol. Med.

Mol. Microbiol.

Enzymatic and nonenzymatic mechanisms for ferric leghemoglobin reduction in legume root nodules

Proc. Natl. Acad. Sci. USA