humanneuroglobinfunctionsasaredox …an applied photophysics sx-20 stopped-flow instrument equipped...

Human Neuroglobin Functions as a Redox-regulated NitriteReductase*□S

Received for publication, July 16, 2010, and in revised form, February 4, 2011 Published, JBC Papers in Press, February 4, 2011, DOI 10.1074/jbc.M110.159541

Mauro Tiso‡1,2, Jesus Tejero‡1, Swati Basu§, Ivan Azarov§, Xunde Wang¶, Virgil Simplaceanu�, Sheila Frizzell‡,Thottala Jayaraman‡, Lisa Geary‡, Calli Shapiro‡, Chien Ho�, Sruti Shiva‡**, Daniel B. Kim-Shapiro§,and Mark T. Gladwin‡ ‡‡3

From the ‡Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15213, the §Department of Physics, WakeForest University, Winston-Salem, North Carolina 27109, the ¶NHLBI, National Institutes of Health, Bethesda, Maryland 20892, the�Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213, the **Department ofPharmacology and Chemical Biology and ‡‡Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh,Pittsburgh, Pennsylvania 15213

Neuroglobin is a highly conserved hemoprotein of uncertainphysiological function that evolved from a common ancestor tohemoglobin and myoglobin. It possesses a six-coordinate hemegeometrywith proximal and distal histidines directly bound to theheme iron, although coordination of the sixth ligand is reversible.Weshowthatdeoxygenatedhumanneuroglobinreactswithnitriteto formnitric oxide (NO). This reaction is regulated by redox-sen-sitive surface thiols, cysteine 55 and46,which regulate the fractionof the five-coordinated heme, nitrite binding, and NO formation.Replacementof thedistal histidineby leucineorglutamine leads toa stable five-coordinated geometry; these neuroglobin mutantsreduce nitrite to NO �2000 times faster than the wild type,whereas mutation of either Cys-55 or Cys-46 to alanine stabilizesthe six-coordinate structure and slows the reaction. Using lentivi-rus expression systems, we show that the nitrite reductase activityof neuroglobin inhibits cellular respiration viaNObinding to cyto-chrome c oxidase and confirm that the six-to-five-coordinate sta-tus of neuroglobin regulates intracellular hypoxic NO-signalingpathways. These studies suggest that neuroglobinmay function asa physiological oxidative stress sensor and a post-translationallyredox-regulated nitrite reductase that generates NO under six-to-five-coordinate heme pocket control.We hypothesize that the six-coordinate heme globin superfamily may subserve a function asprimordial hypoxic and redox-regulatedNO-signaling proteins.

A phylogenic analysis of the heme-globin family indicatesthat thewell characterized proteins hemoglobin andmyoglobin

were antedated by neuroglobin, which already existed 800 mil-lion years ago (1, 2). Neuroglobin (Ngb)4 sequences remainedhighly conserved throughoutmammalian evolution, suggestinga strongly selected vital functionality (3). This heme-contain-ing, monomeric 16.9-kDa protein shares 21 and 25% sequencesimilarity with myoglobin and hemoglobin. However, unlikemyoglobin and hemoglobin, it possesses a bis-histidine six-co-ordinate heme geometry, such that the proximal and distal his-tidines in the heme pocket are directly bonded to the heme iron(both Fe2� or Fe3� oxidation states) (4). Indeed, at equilibriumthe concentration of the five-coordinate neuroglobin is verylow, reported from 0.1 to 5% (5). Binding of oxygen or other gasligands, such as nitric oxide (NO) or carbon monoxide, to theheme iron occurs upon displacement of the 6th coordinationbond with the distal histidine 64 residue (6, 7). Despite thisstructural difference with myoglobin, neuroglobin displayscomparable�-helix globin folding and high oxygen affinity (P50about 1–2 mm Hg at 20 °C) (8, 9). However, the low tissueconcentration of neuroglobin and the rapid auto-oxidation ofthe oxygen-bound species suggest neuroglobin has not evolvedto store and supply oxygen, leading to a number of differenthypotheses about its molecular functionality (2, 10).In vitro and in vivo expression of neuroglobin produces cyto-

protective effects, limiting neuronal cell death during glucosedeprivation and hypoxia and limiting the volume of braininfarction in stroke models (11–14). Understanding the func-tionality of neuroglobin could provide a paradigm shift in bothbiology and therapeutics because several highly conservedheme-globins, ubiquitous in plants and animals, exist in equi-librium between dominant six-coordinate heme geometry anda less frequent five-coordinate state. Examples include cytoglo-bin, cytochrome c, Drosophila melanogaster hemoglobin, andthe nonsymbiotic plant hemoglobins (15–17).Over the past 5 years, our groups have examined the ability of

deoxygenated hemoglobin and myoglobin to react with andreduce nitrite to NO (18, 19). We have proposed that this reac-tion subserves a function similar to the bacterial nitrite reduc-tases, in which a coupled electron and proton transfer to nitritegenerates NO.

* This work was supported, in whole or in part, by National Institutes of HealthGrants HL058091 (to D. B. K.-S.), GM084614 (to C. H.), and HL098032 (toM. T. G.). This work was also supported by Institute for Transfusion Medi-cine, Hemophilia Center of Western Pennsylvania (to M. T. G. and S. S.), andAmerican Heart Association Grant 109SDG2150066 (to S. S.). M. Tiso, M. T.Gladwin, and D. B. Kim-Shapiro are listed as co-inventors on a patent appli-cation entitled “Neuroglobin as a Six-to-Five-coordinate Regulated NitriteReductase.”

□S The on-line version of this article (available at http://www.jbc.org) containsTable S1, Figs. S1–S5, Equations S1–S20, and the detailed derivation ofEquation 1.

1 Both authors contributed equally to this work.2 Present address: Dept. of Chemistry and Biochemistry, University of Mary-

land, College Park, MD 20742.3 To whom correspondence should be addressed. Tel.: 412-692-2210; Fax:

412-692-2260; E-mail: [email protected]. 4 The abbreviations used are: Ngb, neuroglobin; Mb, myoglobin.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 20, pp. 18277–18289, May 20, 2011Printed in the U.S.A.

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In the heart, myoglobin can reduce nitrite to NO to regulatehypoxic mitochondrial respiration and enhance the cellularresilience to prolonged ischemia, analogous to the cytoprotec-tive effects of neuroglobin (19). Studies using the myoglobinknock-out mouse have now confirmed that myoglobin is nec-essary for the following: 1) nitrite-dependent NO and cGMPgeneration in the heart; 2) nitrite-dependent cytoprotectionafter ischemia/reperfusion, and 3) nitrite-dependent control ofhypoxic cellular respiration (20). It is therefore apparent thatbothmyoglobin and neuroglobinmay have roles in limiting celldeath after ischemia-reperfusion injury. Of relevance to neuro-globin, we have recently discovered that themitochondrial pro-tein cytochrome c can reduce nitrite to NO more rapidly thaneither hemoglobin or myoglobin, but only when it assumes thefive-coordinate conformation (21). This conformation onlyoccurs during the interaction with anionic phospholipids orupon oxidation or nitration of protein residues, suggesting apost-translational tertiary structure regulation of nitrite reduc-tion and NO generation.Interestingly, human neuroglobin contains two surface cys-

teines (Cys-46 and Cys-55) that form a disulfide bridge uponoxidation (22). Disulfide bond formation is accompanied by adecrease in the distal histidine binding affinity to heme iron(KHis has been shown to decrease from �3000 to 280, and val-ues are calculated as kon/koff and are dimensionless) (23). Thisin turn increases the subpopulation of five-coordinate neuro-globin and increases the affinity for endogenous ligands such asoxygen (P50 shift from about 9 to 1 mm Hg) (22). Nicolis et al.(24) reported that the oxidized disulfide-bridged neuroglobinalso exhibits a higher affinity for nitrite than the thiol-reducedform.We therefore hypothesized that neuroglobin, and more gen-

erally the six-coordinate heme globins, may function as post-translationally redox-regulated nitrite reductases that generateNOunder control of the six-to-five-coordinate heme iron tran-sition. Such functionality may underlie hypoxic neuroprotec-tive signaling and the control of hypoxic cellular respiration.

EXPERIMENTAL PROCEDURES

Reagents and Standards Sample Preparation—All reagentswere purchased from Sigma unless otherwise specified. UV-visible spectra and kinetic data were recorded on an HP8453UV-visible spectrophotometer (Agilent Technologies, PaloAlto, CA). Superdex S200 gel filtration columns were pur-chased fromGEHealthcare. Solutions of sodiumdithionite andnitrite were prepared with argon-degassed 0.1 M phosphatebuffer, pH 7.4, and kept at 25 °C under inert gas. Purchasedhorse heart myoglobin was further purified by passing througha Sephadex G-25 gel filtration column and eluting with 0.1 M

phosphate buffer, pH 7.4. Neuroglobin was oxidized withexcess potassium ferricyanide or reduced by incubation with500 mM sodium dithionite; excess reagents were removed bypassing the mixture through two sequential Sephadex G-25desalting columns. Met-Ngb concentrations were estimated bymeasuring the absorbance of the heme Soret band using �414 �129 mM�1 cm�1. Standard reference species of recombinantNgb for spectral deconvolutionwere prepared following proce-dures previously described for hemoglobin and myoglobin (18,

19, 25). Reference spectra were recorded for deoxy-Ngb, iron-nitrosyl-Ngb, met-Ngb, and oxy-Ngb. When necessary, anaer-obically reducedNgb samples were prepared in glovebox undera 2–4%H2 atmosphere of catalyst-deoxygenated nitrogen, col-lected directly in cuvettes, and sealed with rubber septa insidethe glovebox. To reduce the intramolecularNgb disulfide bond,Ngb solutions were dialyzed in PBS containing 10 mM DTTdissolved in degassed 100 mM phosphate buffer and 0.5 mM

EDTA as described previously (24).Cloning, Expression, and Purification of Recombinant Ngb—

Molecular biology was performed using standard tech-niques. For the expression of the 151-amino acid polypep-tides of human Ngb, the cDNA SC122910 was cloned inBL21(DE3)pLysS(pET28a). Cells were grown in LB brothcontaining 30 �g/ml kanamycin and 25 �g/ml chloramphen-icol; expression was induced with 1 mM isopropyl 1-thio-�-D-galactopyranoside and carried out for 4 h at 37 °C, including�-aminolevulinic acid (0.4 mM) in the media. Purification wascarried out as described with minor modifications (26). Toincrease purification yield, human Ngb cDNAwas fused with aHis6 tag in the N termini and cloned into Escherichia coliBL21(DE3)pET28a for protein overexpression, and His-taggedhuman Ngb was purified using nickel-nitrilotriacetic acid-aga-rose (Qiagen, Valencia, CA) affinity column according to themanufacturer’s instructions. The eluted protein was dialyzedagainst PBS at 4 °C, concentrated with 10-kDa cutoff filter, andstored in aliquots at �80 °C. The additional amino acids at theN terminus of His-tagged Ngb were removed using a thrombincleavage capture kit (Novagen, Gibbstown, NJ). The purity ofeach recombinant Ngb batch prepared was assessed by SDS-PAGE and UV-visible spectroscopy. The number of accessiblethiol groups per heme was measured by the 4,4�-dithiodipyri-dine assay (27).Mutagenesis of Recombinant Ngb—Site-directed mutagene-

sis was performed using QuikChange II kit (Stratagene, PaloAlto, CA). The oligonucleotides for mutation C46A, C55A,H64L, and H64Q are reported in supplemental Table 1. Thetemplate used forC46A andC55Awas pCMV-1A and forH64Land H64Q was pET28a. Clones were sequenced to confirm thedesired mutations. Expression and purification of mutant Ngbwere carried out using the same procedures as for wild typeNgb.Anaerobic Reactions of Globins with Excess Nitrite—Reaction

kinetics of known amounts of Mb or Ngb with nitrite weremonitored by absorption spectroscopy for the indicated time ina cuvette in the presence or in the absence of 2–4 mM sodiumdithionite. All reactions were run at 25 or 37 °C in 0.1 M phos-phate buffer at controlled pH. Deoxygenated nitrite was added,using an airtight syringe, to a sealed anaerobic cuvette to initiatethe reaction. Oxygen contamination was prevented by applica-tion of positive argon pressurewithout a channel for gas escape.Concentrations of single species during reactions were deter-mined by least squares deconvolution of the visible absorptionspectrum into standard reference spectra usingMicrosoft Excelanalysis. Oxy-Ngb was included to confirm successful deoxy-genation before the reaction. To vary pH values, deoxy-Ngband nitrite were prepared in phosphate buffer adjusted to thetarget pH values. Fast kinetic studies were performed using

Neuroglobin Heme Coordination Regulates Nitrite Reduction

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an Applied Photophysics SX-20 stopped-flow instrumentequipped with rapid-scanning diode array detection (AppliedPhotophysics Ltd., Leatherhead, Surrey, UK). Experimentswere carried out at 25 °C by rapidly mixing a solution ofreduced deoxy-Ngb containing 2 mM dithionite with a knownsolution of nitrite at controlled pH. To determine bimolecularrate constants, all reactions were analyzed with Pro-K software(Applied Photophysics Ltd., Leatherhead, Surrey, UK) usingsingular value decomposition followed by fitting of the reduceddata matrix to a pseudo first-order kinetic model.Model of the Wild Type Human Ngb Structure—Crystalliza-

tion of the wild type humanNgb is hindered by aggregation andprecipitation problems.Mutation of the three cysteine residuesyielded a protein suitable for crystallization studies (28). Thereported structure (ProteinDataBank code 1OJ6) thus includesthe mutations C46G, C55S, and C120S. To assess the possiblestructure of the wild type enzyme, a homology model was builtusing the Swiss-Model server (29) with the sequence of the wildtype Ngb and the available human structure as template. Thecoordinates of the heme molecule were copied from the 1OJ6structure.Determination of the Midpoint Redox Potential of the Thiol/

Disulfide Couple in Ngb—Wild type and C55A mutant Ngb(50–60 �M) were incubated at 37 °C in an anaerobic gloveboxwith solutions containing various ratios of reduced (GSH) andoxidized (GSSG) glutathione, with the total GSH and GSSGconcentration fixed at 20 mM in 0.1 M phosphate buffer, pH 7.0(30). The GSH/GSSG ratio was varied to establish a gradient ofredox potentials between �130 and �250 mV, calculated bythe Nernst equation according to a midpoint reduction poten-tial of �240 mV. After 1 or 2 h of incubation, glutathione wasremoved anaerobically by passage through a G-25 column, andNgb was reacted immediately with 10 mM nitrite in 0.1 M phos-phate buffer, pH 7.0, as described above. The observed rateconstant determined at each glutathione ratio was fitted usingthe Nernst equation, and the midpoint reduction potential ofthe thiol/disulfide couple of Ngb was calculated.Determination of Nitrite Binding Constants—The binding

constant of nitrite tomet-Ngbwas determined by incubation of10 �Mwild type or mutant Ngb with increasing concentrationsof nitrite in 200 mM phosphate buffer, pH 7.4, in a cuvette at25 °C, and the UV-visible spectra were recorded after eachincrease in nitrite concentration. The dissociation constant Kdfor each proteinwas determined by interpolation of the absorb-ance difference data following procedures in Nicolis et al. (24).The reaction rate of nitrite and met-Ngb was then determinedunder the same conditions at 100 mM nitrite.NMRSpectroscopy—1HNMRspectra in 1H2Owere collected

at 29 °C on a Bruker DRX-600 NMR spectrometer (Bruker, Bil-lerica, MA) operating at 599.79 MHz with a 5-mm triple reso-nance probe using a water presaturation pulse sequence with1-s irradiation time. Samples of wild type and mutant 250–300�M met-Ngb were prepared in 0.1 M phosphate buffer, pH 7.4.Typically, 1024 transientswere averaged, using 90° pulses, spec-tral width of 80 ppm, and 16 K time domain points. Spectra arereferenced indirectly through the resonance of thewater, whichoccurs at 4.76 ppm downfield from the methyl resonance of2,2-dimethyl-2-silapentane-5-sulfonate.

Electron Paramagnetic Resonance Spectroscopy—Ironnitrosyl species were measured by EPR spectroscopy using aBruker EMX 10/12 spectrometer (Bruker, Billerica, MA) oper-ating at 9.4 GHz, 5-G modulation, 10.1-milliwatt power,327.68-ms time constant, and 163.84-s scan over 600G at 110Kas described previously (21, 31). The concentrations of Mb andNgb species were determined by performing the double inte-gral calculation and comparing with standard samples.Direct Measurement of NO Release by Chemiluminescence—

Deoxy-Ngb (final concentration 20 �M) was injected in a reac-tion vessel containing 100 mM phosphate buffer, pH 7.4 (3 ml),pre-equilibrated, and purgedwith helium and connected in lineto a nitric oxide analyzer (NOA 280i) (Sievers, GE AnalyticalInstruments, Boulder, CO). Once a stable base line was estab-lished, Ngb was reacted with a known amount of nitrite asdescribed previously (18). Traces were smoothed by a runningaverage spanning 2 s.Respiration of Isolated Mitochondria in the Presence of Ngb—

Mitochondria were isolated from the livers of male Sprague-Dawley rats and incubated with wild type or mutant Ngb pro-teins in a sealed, stirred chamber at 37 °C. State 3 respirationwas stimulated with succinate (15 mM) and ADP (1 mM), andoxygen consumption was measured with a Clark-type oxygenelectrode. All experiments with respiring mitochondria wereperformed according to previously reported procedures (19).Similar experiments were performed with SH-SY5Y cells sus-pended in the respirometer and treatedwith the uncoupler car-bonyl cyanide p-trifluoromethoxyphenylhydrazone (5 �M) tomeasure hypoxic inhibition of cellular respiration.Immunoblotting of Ngb Expression in SH-SY5Y Neuronal

Cells—Equal amounts of denatured total proteins (25 �g) fromthe SH-SY5Y neuronal cells expressing GFP vector, wild typeand H64L mutant Ngb, were subjected to 4–15% SDS-poly-acrylamide gradient gels and immunoblotted with anti-GFPmonoclonal antibody (Santa Cruz Biotechnologies, Inc., SantaCruz, CA) and then scanned using a Odyssey imaging system(LI-COR Biosciences, Lincoln, NE).Statistical Analysis—Each experiment was performed at

least in triplicate, and values are representative of two ormore independent determinations using different batches ofprotein purified separately. Data were analyzed using Origin8.0 (OriginLab Corp., Northampton, MA) and expressed asmean � S.D. Analysis for statistically significant differencesamong mean values was done, when applicable (Figs. 2B, 5B,and 6B), using the one-way analysis of variance.

RESULTS

Nitrite Is Reduced to NO via Reaction with DeoxygenatedHuman Neuroglobin—To examine the reaction of nitrite withneuroglobin, we expressed and purified recombinant humanneuroglobin. Spectrophotometric analysis of our proteins con-firmed the six-coordinate heme structure in both the ferrousand ferric states of Ngb, with visible �- and �-peaks around the550 nm wavelength (supplemental Fig. 1A). We prepared fer-rous deoxy-Ngb in an anaerobic glovebox as detailed under“Experimental Procedures” and recorded the visible spectra ofthe reaction between 10 �M deoxy-Ngb and 10 mM nitrite at25 °C at constant intervals in a sealed air-tight cuvette under




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external argon pressure (Fig. 1A). The time-dependent changesof deoxy-Ngb, ferric met-Ngb, and iron-nitrosyl-Ngb (Fe2�-NO) species (Fig. 1B) were calculated by least squares deconvo-lution of the reaction spectra using standard reference spectra(supplemental Fig. 1A). In an anaerobic environment, nitrite isreduced to NO according to Reaction 1, and the NO generatedhas very high affinity for the ferrous Ngb heme (Fe2�) (WTNgb: kon � 1.5 � 108 M�1 s�1 and koff � 2 � 10�4 s�1; H64LNgb: kon � 2.7 � 108 M�1s�1 and koff � 2 � 10�4 s�1; H64QNgb: kon � 1.9� 108 M�1 s�1 and koff � 3� 10�4 s�1(32)) thusyielding ferric Ngb heme (Fe3�) and iron-nitrosyl-heme (Fe2�-NO) as a final reaction product (Reaction 2).

Fe2� � NO2� � H�3 Fe3� � NO� � OH�

REACTION 1

NO� � Fe2�3 Fe2� � NOREACTION 2

Weobserved a reaction stoichiometry consistentwith the reac-tionofnitritewithhemoglobinormyoglobin,with twodeoxy-Ngbmolecules forming one iron-nitrosyl-Ngb and one ferricNgb (Fig.1B). Analysis of the bimolecular rate constant indicated that thereactionofnitritewithNgbproceedsoverall at0.12�0.02M�1 s�1

at 25 °C, pH 7.4 (0.26� 0.02 M�1s�1 at 37 °C). A recent study (33)reported that the reactionofdeoxymouseneuroglobinwithnitritein the range 7–230 �M generated ferric met-Ngb in excess of fer-rous nitrosyl-Ngb at apparent second-order rate constant of 5.1�0.4 M�1 s�1; however, our experimental conditions with humanneuroglobin differ considerably.

Both Gladwin and co-workers (34) and Salhany (35) haveshown that the reaction of nitrite with hemoglobin in the pres-ence of dithionite proceeds via Reactions 1 and 2, but the ferricheme that is formed is reduced back to the ferrous form tocontinue the reaction. Thus, iron-nitrosyl-heme forms at thesame rate as deoxyheme is consumed, and the overall stoichi-ometry is one deoxy-Ngb forming one iron-nitrosyl-Ngb. Per-forming the reaction in the presence of dithionite limits theauto-oxidation of the ferrous heme prior to the reaction withnitrite and allows for facile assessments of anaerobic reactionmechanisms and kinetics. By complementary studies using myo-globin, we verified that the rate-limiting step of the reaction in thepresence of dithionite is the heme iron catalyzed conversion ofnitrite to NO (supplemental Fig. 2, A and B). We then performedthe reaction of anaerobic nitrite and deoxy-Ngb (10 mM and 10�M,respectively)asdescribedabove in thepresenceof3mMexcessdithionite at pH 7.4 in 100mM phosphate buffer (Fig. 1,C andD).The stoichiometry was consistent with one deoxy-Ngb formingone iron-nitrosyl-Ngb, and the calculated bimolecular rate con-stant was 0.11 � 0.01 M�1 s�1, in accordance with the valueobtained in the absence of dithionite.We further investigated thereactivity of deoxy-Ngb with nitrite in the concentration range0.25–20mM(Fig. 1E).Thesecond-orderbimolecular rateconstantderived from the linear fit of the observed rate constants versusnitrite concentration is 0.12� 0.02M�1 s�1 in agreementwith thecalculated instantaneous reaction rate.Proton Dependence of the Nitrite Reductase Reaction with

Neuroglobin—We next explored whether deoxy-Ngb-depen-dent nitrite reduction requires a proton (Reaction 1).We deter-

FIGURE 1. Anaerobic reaction of deoxyneuroglobin with nitrite in the absence and presence of dithionite. A, selected visible spectra of the reactionbetween 10 �M deoxy-Ngb and 10 mM nitrite at 1-min intervals. B, time-dependent changes of deoxy-Ngb (blue), iron-nitrosyl-Ngb (green), and total met-Ngb(red) concentration during the reaction. C and D, as in A and B, respectively, for the reaction in the presence of 3 mM dithionite. E, plot of observed rate constants(kobs) versus nitrite concentration; the second-order bimolecular rate constant obtained from the linear fit of the data is 0.12 � 0.02 M

�1 s�1. F, effect of pH onthe nitrite reductase reaction rates. Inset, bimolecular rate constant is linear with the proton concentration, and it extends through the zero point (line showslinear regression analysis of the data). All measurements were made in 100 mM phosphate buffer and at 25 °C as described under “Experimental Procedures.”




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mined the pH dependence of the bimolecular rate constant ofthe nitrite reductase reaction near the physiological range, pH6.5–8.0 (Fig. 1F). We found that increasing concentrations ofprotons accelerates the reaction rate by 10-fold for each pHunit decrease. The slope of the linear fit, which representsthe order of rate dependence on [H�], is 0.96, close to theideal of 1.0, and it extends through the zero point (Fig. 1F,inset) indicating the requirement for one proton in the reac-tion. We conclude that the reaction constitutes a concertedelectron and proton transfer to nitrite to form NO analogousto bacterial nitrite reductase.Surface Cysteines Cys-46 and Cys-55 Regulate the Heme Pocket

Coordination and the Rate of Nitrite Reduction to NO—Unlike most other globins, human Ngb displays three con-served cysteines (notable exception being mouse Ngb) at posi-tions 46, 55, and 120 located on the protein surface as shown inthe wild type thiol-reduced human Ngb structure model (Fig.2A). When oxidized, cysteines 46 and 55 form an intramolecu-lar disulfide bond (36), which influences the position of theE-helix containing the distal histidine (22) and regulates theheme ligand binding equilibrium. Reduction of the disulfidebond allows additional structural freedom in the orientation ofthe E-helix (Fig. 2A), which leads to an increased proportion ofmolecules in the six-coordinate state and thus reduced oxygenand nitrite binding affinities (22, 24). We determined by the4,4�-dithiodipyridine assay the number of accessible thiols perheme in our wild type Ngb, as purified, reduced by DTT, and in

the Cys-55 to alanine mutant Ngb (Fig. 2B). The results areconsistent with the quantitative formation of a disulfide bondduring protein purification and the presence of the singlereduced Cys-120 in the oxidized thiol form. To determinewhether the rate of nitrite reduction is influenced by the redoxstate of cysteines 46 and 55, we first reduced the cysteines byincubation with 10 mM DTT and then measured the rate ofnitrite reduction after anaerobic DTT removal. Fig. 2C showsthat reduction of the disulfide bond slows down the rate byabout 2-fold (0.062 � 0.005 M�1 s�1 at 25 °C, pH 7.4). Todirectly test the hypothesis that disulfide bridge reductionaffects the nitrite reactivity of neuroglobin, we generatedrecombinant mutants with cysteine 55 or 46 replaced by ala-nine (C55A and C46A), which slowed down the rate of nitritereduction to similar rates observed with Ngb having fullyreduced cysteines (Fig. 2C). For a direct comparison, we reportthe bimolecular reaction rates for WT and mutant Ngb pro-teins in Table 1 together with values for myoglobin andhemoglobin.Physiological Redox Control of the Cys-46 to Cys-55 Disulfide

Bond Regulates the Rate of Nitrite Reduction to NO—We nextdetermined if the formation of a disulfide bond betweenCys-46and Cys-55 is redox-regulated within the physiological range ofcellular redox state. We incubated wild type and C55A Ngbwith increasing ratios of reduced/oxidized glutathione thatestablished a gradient of ambient redox potentials (37). After120 min of incubation, we removed glutathione anaerobically

FIGURE 2. Redox state of cysteines 46 and 55 modulates nitrite reductase reactivity. A, model of the wild type human neuroglobin structure with theindicated reduced cysteines Cys-46, Cys-55, and Cys-120. B, determination of the number of reduced cysteines by the 4,4�-dithiodipyridine assay (see “Exper-imental Procedures.”). C, comparison of the decrease of deoxy-Ngb and the formation of iron-nitrosyl Ngb over time for wild type Ngb with oxidized (SS) andreduced (SH) thiol and C46A and C55A mutant Ngb. D, observed nitrite reductase rate constants versus determined redox potentials. Data were fit using theNernst equation. The midpoint redox potential of the thiol/disulfide couple in wild type Ngb is �194 � 3 mV. SHE, standard hydrogen electrode. E, comparisonof the NMR spectrum of wild type and C55A mutant met-Ngb. F, nitrite binding affinity constant determination by difference spectral titration for wild type,DTT-reduced, and C55A mutant Ngb by differential spectra after indicated nitrite addition.




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by passage through a G-25 column and measured the rates ofnitrite reduction (Fig. 2D). We found that there was a suddenand substantial drop in the observed nitrite reductase rate con-stants (kobs) with decreasing redox potential only for the wildtype protein. Fitting the data to the Nernst equation provided amidpoint reduction potential of the Cys-46/Cys-55 thiol/disul-fide redox couple of �194 � 3 mV. This value is within therange of cellular redox potentials (37).To directly examine whether the cysteines redox state causes

changes in heme pocket molecular and electronic structure, wecompared the NMR spectrum of wild type and C55A mutantmet-Ngb (Fig. 2E). Characteristic NMR signals for the hememethyls are visible in the spectral regions around 36 and 23ppm and 20 to 12 ppm and were assigned by comparisonwith the published spectra (38, 39). The two spectra arelargely similar, but a few marked differences in the positionsof several hememethyl resonances (M8-B, M5-A,M1-A, andM5-B) as well of several hyperfine shifted resonancesbetween 18 and 12 ppm (region marked with an asterisk inFig. 2E) are evident. Also, some unassigned ring currentsshifted resonances around �2 ppm are different. We con-clude that the thiol mutation C55A affects the geometry ofthe heme pocket environment.Nicolis et al. (24) reported that the oxidized disulfide-

bridged (SS) met-Ngb exhibits a higher affinity for nitrite thanthe thiol-reduced (SH) form. We then determined the nitriteaffinity for the wild type SS- and SH-Ngb and for C55Amutantmet-Ngb by difference spectra titration (Fig. 2F). Because of thesmall absorbance changes, the calculated dissociation con-stants (Kd), reported in Table 2, show large standard deviations;nevertheless, there is an apparent influence of the redox state ofthe cysteines on the distal histidine and nitrite affinity to theheme iron. During these experiments, we noticed thatmet-Ngbvery slowly reacts with nitrite to produce nitrosyl-Ngb (bimo-lecular rate constants reported in Table 2). The slow rates ofreaction produce a detectable spectroscopic effect only at highnitrite concentrations, approaching 0.1 M, and result in an arti-ficial decrease of maximal absorbance difference that has pre-

viously been assigned to a second low affinity binding constant(24). These experiments indicate that the redox state of cys-teines 46 and 55 regulates both the five-to-six-coordinate equi-librium and the rate of nitrite conversion to NO. Intriguingly,an analogous effect is observed with hemoglobin, in which oxi-dation of cysteine 93 speeds up the rate of nitrite reduction toNO, and reduction slows the rate (40). This effect has beenattributed to the effect of thiol oxidation on decreasing theheme redox potential.Rate of Nitrite Reduction Is Maximal in the Five-coordinate

State of Neuroglobin—To test the hypothesis that a change inthe equilibrium between the five- and six-coordinate Ngb sub-populations mediates the control of the nitrite reduction rate,we generated recombinantNgbswithHis-64 replaced by Leu orGln. The absorbance spectra analysis of oxygenated and deox-ygenated ferrous H64L and H64Q Ngb and the ferric species(supplemental Fig. 1) confirmed that bothmutants are “locked”in the five-coordinate conformation (41) and have very similarspectral characteristics to the classic five-coordinate heme pro-tein myoglobin (supplemental Fig. 1). We examined the reac-tion of nitrite with deoxygenated H64L Ngb in the presence ofexcess dithionite similarly to experiments with wild type Ngbbut using 100 �M nitrite (Fig. 3, A and B). To our surprise, therate of deoxy-Ngb conversion to nitrosyl-Ngb was extremelyfast, and the bimolecular rate constant was �2000-fold higherthan for the wild type Ngb. We then used fast mixing stopped-flow spectroscopy to determine the rates of the reaction in therange 10–1000 �M nitrite (Fig. 3C). The observed rate con-stants increased linearly with increasing nitrite concentrations,and the bimolecular rate constant derived from the linear leastsquare fit was 259 � 8 M�1 s�1 at 25 °C, pH 7.4. Examination ofthe reaction at different pH values (Fig. 3D) indicates that thereaction requires a proton similar to the reaction with wild typeNgb. Remarkably, the rate increases above 2,500 M�1 s�1 at pH6.5 and 25 °C. The H64Q mutant showed a similar behavior,with a rate constant of 267� 16 M�1 s�1 at 25 °C, pH 7.4 (Table1, supplemental Fig. 3), and rate constants above 2,000 M�1 s�1

at pH 6.5. These are the fastest reactions of nitrite with a heme-globin ever reported and confirm our hypothesis that the six-to-five-coordinate heme pocket transition regulates the rate ofnitrite reduction to NO.We next determined the nitrite binding affinity forH64L and

H64Q met-Ngb by fitting of the maximal changes in the Soretband as a function of nitrite concentration (Fig. 3E, normalizeddifference spectra titration compared with wild type SS-Ngb).The calculatedKd values for thesemutants are comparablewiththewild typeNgb (Table 2); however, the totalmaximal absorb-ance difference for H64L and H64Q mutants is more than

TABLE 1Summary of bimolecular reaction rates for the reaction of heme-con-taining deoxyglobins with nitrite (Hb, human hemoglobin; sw Mb,sperm whale myoglobin; Ngb, neuroglobin)All reactions were studied in 100mM sodiumphosphate buffer, pH 7.4. Hemoglobinvalues were determined at 37 °C. Myoglobin and neuroglobin values were deter-mined at 25 °C.

Protein k

M �1 s�1

Hb (T state) �0.12aHb (R state) �6aHorse Mb 2.9 � 0.2sw MbWT 5.6 � 0.6sw Mb H64A 1.8 � 0.3sw Mb H64L Very slowb

NgbWT SS 0.12 � 0.02Ngb WT SH 0.062 � 0.005Ngb C55A 0.060 � 0.008Ngb C46A 0.058 � 0.006Ngb H64L 259 � 8Ngb H64Q 267 � 16

a Values are from Ref. 18.b The reaction of Mb H64L is significantly (more than 10-fold) slower than wildtype or H64A sperm whale Mb and apparently independent of NO2

� in theconcentration range studied (1–5 mM).

TABLE 2Nitrite dissociation constants (Kd) and bimolecular rate constants (k)for reactions of met-Ngb with nitrite determined at 25 °C in 200 mM

phosphate buffer, pH 7.4Protein Kd (NO2

�) Rate of nitrite ferric heme reduction

mM M �1 s�1

NgbWT SS 6.2 � 2.1 0.0005 � 0.0005Ngb WT SH 12.6 � 3.3 0.0002 � 0.0005Ngb C55A 30.1 � 4.5 0.0002 � 0.0004Ngb H64L 8.3 � 1.8 0.032 � 0.002Ngb H64Q 12.5 � 1.8 0.016 � 0.009




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20-fold greater than wild type (supplemental Fig. 4). Theseresults suggest that in wild type Ngb His-64 binding may out-compete nitrite binding even at high concentrations so only the�1% of five-coordinatemoleculesmay bind nitrite, whereas forH64L andH64Qa fully bound situation is possible. The spectralchanges for H64L and H64Q also indicate differences in thenitrite binding modes. For the H64L mutant, there is a largeincrease in the Soret peak and little change in the 500–600 nmregion. In the case of H64Q there is a decrease in the Soret peakaccompanied by a shift to longer wavelengths and also changesin the 500–600 nm region that resemble the nitrite binding toHb or Mb (supplemental Fig. 4).Finally, we compared the reaction of 1 mM nitrite with our

wild type Ngb (with oxidized and reduced cysteines), mutantH64L, H64Q, and C55ANgb (absorbance decrease of the Soretpeak at 425nm) in 0.1MHEPES, pH7.4. The relative percentageof the total absorbance change occurring in the first 60 min ofthe reaction is shown in Fig. 3F (with H64L or H64Q Ngb nor-malized to 100%, wild type SS-Ngb 38%, wild type SH-Ngb 20%,and C55A Ngb 18%). For both H64L and H64Q Ngb, the reac-tion of the five-coordinate mutant proteins reached the endpoint in the first minute of the reaction and are expanded in theinset of Fig. 3F.Confirmation of Reaction Kinetics Using Electron Paramag-

netic Resonance Spectrometry—EPR spectrometry allows fordirect measurement of the paramagnetic NO-heme (iron-ni-trosyl) species and provides confirmation ofNO formation dur-ing the reaction of nitrite withNgb.We evaluated the Fe2�-NO

build up following reaction of 1 mM nitrite with wild type SS-Ngb, SH-Ngb, and mutants H64L and H64Q Ngb (40 � 5 �M)and compared it with the rate of iron-nitrosyl-myoglobin for-mation (Fig. 4, A and B). EPR spectra analysis confirmed thatthe reduction of the cysteines (stabilizing the six-coordinateheme geometry) slowed the rate of iron-nitrosyl-Ngb forma-tion, although replacement of the distal histidine with leu-cine (five-coordinate stabilization) dramatically increasedthe rate of NO formation. In particular, experiments usingH64L and H64Q Ngb mutants and 1 mM nitrite were almostcomplete in 1 min, and to allow assessment of the reactionkinetics, lower concentrations of Ngb (10 �M) and nitrite (50�M) were necessary (Fig. 4, C and D). The calculated rates ofnitrosyl-Ngb formation are similar to data obtained byabsorbance spectrometry.Nitrite Reduction by Deoxyneuroglobin Generates NO—The

reaction of nitrite with deoxy-Ngb generates NO and ferricNgb. Although in our in vitro conditions deoxy-Ngb can recap-ture the NO, we next explored if free NO gas can escape atmeasurable rates.Wemixed anaerobic Ngb (20 �M) and nitrite(1 mM) in a vessel purged with helium and carried in-line to achemiluminescentNO analyzer. In these conditions, the anaer-obic mixture generated NO in gas phase (Fig. 5A), and the rateof NO formation was again regulated by the cysteines 46–55disulfide bond and by the heme pocket six-to-five-coordinationequilibrium. Fig. 5B shows that the rate of NO detected wassignificantly decreased in reactions with six-coordinate C55ANgb and increased in reactions with the five-coordinate H64L

FIGURE 3. Kinetics of nitrite reaction with mutant H64L Ngb. A and B, spectrophotometric analysis of the anaerobic reaction of 10 �M H64L deoxy-Ngb with100 �M nitrite, pH 7.4, at 25 °C and 3 mM dithionite. C, plot of kobs versus nitrite concentration (10 �M to 1 mM) for H64L Ngb-mediated reduction of nitrite andformation of Ngb Fe2�-NO at pH 7.4 and at 25 °C. The bimolecular rate constant derived from the linear fit of the data is 259 � 8 M

�1 s�1. D, effect of differentpH values on the nitrite reductase rates. Inset, bimolecular reaction rate is linear with the proton concentration. E, nitrite binding affinity constant determina-tion for H64L and H64Q. F, comparison of representative traces of Ngb wild type (with reduced and oxidized surface thiols) and mutants H64L and H64Q. Theabsorbance decreases of the Soret peak (425 nm) are plotted as the percentage of the total absorbance change for human Ngb H64L measured at 25 °C, pH 7.4.




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Ngb, consistent with the hypothesis of six-to-five-coordinateheme pocket control of nitrite reduction. Finally, we incubatedmutant H64L Ngb (30 �M) with increasing amounts of nitrite,startingwith physiologically relevant concentrations (10 and 25�M) up to 1 mM, and we observed a rapid NO generationresponse roughly proportional to the nitrite concentrationinjected (Fig. 5C).Comparison ofMutantMyoglobin andNeuroglobinConfirms

Unique Fast Reactivity of Five-coordinate Neuroglobin withNitrite—To elucidate the factors that confer increased nitritereductase activity to the Ngb H64L mutant, we characterizedthe nitrite reductase activities of Mb mutants H64L and H64Aand Ngb mutant H64Q (summarized in Table 1). Despite thestructural similarities between the two globins, the difference inreactivity for comparable His-64 mutations took oppositedirections. Replacement of Mb His-64 with Leu or Ala leads todecreased reactivity. This may be related to His-64 stabilizingthe heme ligands through hydrogen bonding as highlighted byprevious reports (42, 43). However, Ngb H64Q shows similarrates to theH64Lmutant. This indicates that the removal of the6th ligand and formation of a stable five-coordinate neuroglo-bin is themajor determinant of the increased reactivity, and theresidue polarity constitutes a lesser effect. Possible explana-tions for these effects are discussed below.Nitrite Reduction by Deoxyneuroglobin Mediates NO Signal-

ing—To test whether Ngb-generated NO inhibits mitochondrialrespiration during hypoxia, isolated rat liver mitochondria wereplaced in a sealed, stirred respirometer, and substrateswere addedto stimulate respiration as described previously (19). Mitochon-dria were allowed to respire until the ambient oxygen tensiondropped below detection levels. At this point the respirometer isopened to air oxygen, and cyanide is added to evaluate the time tocomplete inhibition of respiration, as determined by the increase

in oxygen tensions measured with a Clark-type oxygen electrode(Fig. 6A). The extent of mitochondrial inhibition for all experi-ments (without cyanide) is then compared with the effect of cya-nide. We detected no significant inhibition of respiration whennitrite (20 �M) or purified wild type Ngb (5 �M) alone were incu-batedwith respiringmitochondria.However, when the same con-centrations of nitrite and protein were allowed to react together,we observed 78 � 6% inhibition of respiration. As expected, theextent of inhibition was increased significantly by the H64Lmutant Ngb (96 � 2% inhibition) and decreased by the C55AmutantNgb (62�4% inhibition) (Fig. 6B).Toevaluate this incells,the neuronal cell line SH-SY5Ywas stably transfected using a len-tivirus vector with GFP-tagged wild type and H64L mutant Ngb(Fig. 6C) and was used to perform similar experiments. One mil-lion intact SH-SY5Ycellswere suspended in the respirometer, andmaximal respiration ratewas stimulated by addition of the uncou-pler carbonyl cyanide p-trifluoromethoxyphenylhydrazone. Thennitritewas added to cells transfectedwithGFPonly (negative con-trol) and cells expressing wild type Ngb or the H64Lmutant Ngb.In Fig. 6D, we compare the extent of respiration inhibition to thecyanide effect (complete inhibition); cells withGFPonly exhibitedno significant inhibition, but we observed about 15 and 40% inhi-bition, respectively, for wild type and H64LNgb.

DISCUSSION

Altogether, our experiments reveal the following: 1) neuro-globin can function as a nitrite reductase, producing NO fromnitrite; 2) the redox state of the surface thiols of Cys-46 andCys-55 modulates the heme coordination, nitrite affinity, andNO generation and signaling; 3) replacement of the His-64 sidechain locks the heme in a five-coordinate geometry withincreased reactivity toward nitrite; and 4) under hypoxic con-

FIGURE 4. EPR spectroscopy. A and C, EPR spectra showing Fe2�-NO build up following addition of amount of nitrite. B and D, rate of formation of iron-nitrosyl-heme(Fe2�-NO) species measured by EPR. The concentrations were determined by performing the double integral calculation and comparing with standard samples.




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ditions neuroglobin can inhibit mitochondrial respiration inthe presence of nitrite.We also show that the reaction of deoxy-neuroglobin with

nitrite proceeds with stoichiometry formally similar to thereaction of myoglobin or hemoglobin (44). However, in neuro-globin, the process is further regulated by the six-to-five-coor-dinate equilibrium.Amechanistic proposal is presented inScheme1where the first

step of the reaction is the dissociation of His-64 from the hemeiron. The results obtained with Ngb H64L and H64Q mutantsindicate that this process directly limits the nitrite reduction rate.The five-coordinatehemecannowbind toexternal ligands suchasnitrite.Asevidenced in recent reports (45, 46)byexperimental and

density functional theoretical studies, nitritemaybind to thehemein either N-nitro- or O-nitrito-conformations (Scheme 1, secondstep).Wecannot conclude fromour results if either bindingmodeis preferred, and given the differences in the heme pocket of Ngb,as compared with Mb (supplemental Fig. 5), the observationsmade inMbmay not extrapolate (47). Addition of a proton to thenitrite yields the nitrous acid-bound species and those can bedirectly formed by nitrite at low pH. An alternative possibility isindeed the initial binding of HONO to the five-coordinate hemeiron. In the N-bound route, loss of OH� and electron transferyields the Fe3�-NO species with subsequent NO dissociation. Inthe O-bound route, NO dissociates from the bound nitrous acid,and Fe3�-OH� is formed. This met-hydroxide complex can bethenprotonated to the aquomet form. Independently of the route,the end products of the reaction are eventually the same, Fe3�

heme and NO. Interestingly, the dependence of this reaction onthe concentration of proton is opposite to that of the reductivenitrosylation reaction, where the reaction rate increases with theconcentration of OH� (48). These data show that the nitritereductase reaction is the reverse of reductive nitrosylation. FromScheme 1, the relationship between both reactions is apparent,although itmustbenoted that theN- andO-boundroutes indicatetwo different reductive nitrosylation reactions, one involvingFe3�-NO � OH� and another with Fe3�-OH� � NO. It is con-ceivable that depending on the protein (and the concentrations ofNO andOH�), one routemay bemore important than the other.

In myoglobin, the H64L mutation severely impairs ligandbinding of cyanide and azide (42, 49) as hydrogen bondingappears to be necessary to stabilize the ligand. The situation forneuroglobin appears to be very different, and once the His-64-heme interaction is disrupted, the ligand affinity increases. NgbH64L and H46Q show no significant differences in the nitritereduction rates, and thus nitrite reduction appears to be unre-lated to hydrogen bonding.We speculate that the differences inthe nitrite reactivity for Ngb and Mb/Hb (and the differentrequirements for ligand hydrogen binding) can be related todifferences in the preferred nitrite-bindingmode. This, in turn,may be related to the electronic properties of the heme. Theseconcepts deserve further research; resolving the crystal struc-ture of the nitrite-bound ferric Ngb complex could providedefinitive experimental insight into the nature of this rathercomplex nitrite-heme interaction.Modeling of Nitrite Reduction by Five-coordinate Neu-

roglobin—Taking into account the proposed mechanism ofreaction in Scheme 1, we can expand and rewrite our reactionmodel as shown in Reactions 3–7,

Fe2�(6c)7 Fe2�(5c)

REACTION 3

H� � NO2�7 HNO2

REACTION 4

Fe2�(5c) � HNO23 Fe3� � NO� � OH�

REACTION 5

Fe2�(5c) � NO2� � H�3 Fe3� � NO� � OH�

REACTION 6

FIGURE 5. Nitrite reduction by deoxyneuroglobin generates NO gas.A, representative chemiluminescence traces of NO detection in gas phasereleased during the anaerobic reaction of nitrite with buffer only (blue) or 20 �M

deoxy-Ngb wild type (black), H64L (red) or C55A (green). B, quantification of therate of NO detected per min. C, nitric oxide signal measured during incubation of30 �M H64L deoxy-Ngb and increasing concentrations of nitrite.




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NO� � Fe2�3 Fe2� � NOREACTION 7

Then the rate of reduction of nitrite by Ngb can be written asshown in Equation 1,

�dNgb]

dt�

k0H�]�Ka � k�

�1 � KHis)[Ngb][nitrite] (Eq. 1)

where KHis is the ratio of hexacoordinate to pentacoordinateNgb (Reaction 3);Ka is the equilibriumconstant for nitrous acid

FIGURE 6. Deoxyneuroglobin nitrite reduction mediates intracellular NO signaling. A, traces of oxygen consumption by isolated mitochondria showingnitrite-dependent inhibition of respiration; the early rise in oxygen tension indicates NO-dependent inhibition of cellular respiration, which is maximal forcyanide. B, comparison of percentage of extent of inhibition (cyanide defined as 100% inhibition) as measured in A for isolated mitochondria. C, quantificationof expression of GFP only, wild type Ngb, and H64L mutant Ngb in lentivirus-transfected and cloned SH-SY5Y cells by Western blot of 4 –15% SDS-polyacryl-amide gradient gel. D, mean extent of hypoxic inhibition of cellular respiration by incubation of SH-SY5Y cells expressing GFP, wild type Ngb, or H64L Ngb with20 �M nitrite (*, p � 0.01; **, p � 0.001, compared with control).

SCHEME 1. Mechanistic proposal for the reaction of neuroglobin with nitrite (N-bound versus O-bound routes). The first step of the reaction is thedissociation of His-64 from the heme iron (left). The five-coordinate heme can now bind nitrite. Two binding modes for nitrite are possible, N-binding (top rightsection) or O-binding (bottom right section). Subsequent addition of a proton yields the nitrous acid-bound species (those can be directly formed by nitrite atlow pH). In the N-bound route, loss of OH� and electron transfer yields the Fe3�-NO species with subsequent NO dissociation. In the O-bound route, NOdissociates from the bound nitrous acid, and Fe3�-OH� is formed. This met-hydroxide complex can be then protonated to the aquomet form.




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and nitrite anion (about 10�3.15 M; Reaction 4); k0 is the rate ofreaction of nitrous acid with Ngb (Reaction 5), and k� is the ratethat nitrite anion reacts with Ngb (Reaction 6). Equation 1 fol-lows that used byDoyle for the reduction of nitrite by hemoglo-bin (50), combined with the idea that the hememust be at leasttransiently pentacoordinate for the reaction to occur. It

describes the reaction to be bimolecular in nitrite andNgbwiththe bimolecular rate constant k being equal to the term multi-plying the concentration of these reactants;

k �k0H�]�Ka � k�

�1 � KHis)(Eq. 2)

The rate of the reaction of Ngb with nitrite anion, like Hb, issmall compared with that with nitrous acid except at veryhigh pH. In principle, k0 could be different for the WT andH64L or other Ngb mutants. However, if the only or domi-nating effect of the mutation is the formation of a perma-nently pentacoordinate heme (KHis � 0), then one wouldexpect k0 to be the same for both species. However, if themutation affects more than the heme coordination state(such as hydrophobicity in the heme pocket) then we wouldexpect it to change k0 as well.

To test the simple model and determine whether the H64Lmutation does more than affect the coordination of the heme,wemodeled our bimolecular rate constants as a function of pH.Fig. 7A shows the data and fit for the H64L mutant, where KHisis taken as zero. The least squares fit gave k0 as 6.5 � 106 M�1

s�1 and k� as 49 M�1 s�1, confirming that k0 dominates. Next,we used these values to fit the WT data (Fig. 7B), with the onlyfree parameter now beingKHis, which we found to be 4200. Themodel fits the data very well, and the value obtained for KHis isvery much in agreement with those determined previously (forexample 3000 (8)). Additional calculations with the H64Qmutant (supplemental Fig. 3) yielded values of k0 as 4.6 � 106M�1 s�1 and k� as 50 M�1 s�1, in remarkable agreement giventhe values determined for H64L. In conclusion, our results sup-port the idea that the rate differences between WT and H64L/H64QNgb can be primarily determined by the heme coordina-tion (i.e. the six-coordinated to five-coordinated transition),and the nitrite reactivity of the WT enzyme could be vastlyincreased by changes in the KHis parameter.Physiological Relevance of the Nitrite Reductase Activity of

Neuroglobin—Neuroglobin has been shown to promote sur-vival of neurons in hypoxic conditions, but its physiologicalfunction is still unclear. Someof the proposed functions includeoxygen supply, reactive oxygen species/reactive nitrogen spe-

FIGURE 7. Fitting of bimolecular rate constants as a function of pH. Equa-tion 2 was used to fit the data with Ka taken as 7 � 10�4

M�1. A, data for H64L

were fit allowing k0 and k� to vary. B, data for WT were fit allowing only KHis tovary using the values obtained from fitting of H64L data (A) for the otherparameters.

FIGURE 8. Neuroglobin act as a nitrite reductase under oxidative stress conditions. In normal conditions, cells keep a high concentration of reducedglutathione (GSH) and low oxidized glutathione (GSSG). In these circumstances, the disulfide bond of Ngb is not formed, and the protein has a low nitritereductase activity (left). As oxidative stress conditions develop (right), reduced glutathione is consumed, and the number of neuroglobin molecules withformed disulfide bonds increases. The production of NO from nitrite increases, causing the inhibition of respiratory enzymes and limiting oxygen consumptionand reactive oxygen species-producing reactions.




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cies detoxification, regulation of signaling pathways (10), andmore recently, inhibition of cytochrome c-induced apoptosis(52). Ngb is expressed in metabolically active cells and organs(neurons, endocrine organs, retina, etc.) and has been hypoth-esized to interact with mitochondria and mediate cytoprotec-tive responses to ischemic stress (53). NO binding to cyto-chrome c oxidase has been shown to reversibly inhibit electrontransport at low oxygen tensions, in a process thought to con-tribute physiologically to hypoxic vasodilation and to the exten-sion of oxygen diffusion gradients (54, 55). There is alsoincreasing evidence of neuroprotective effects of NO andnitrite/nitrate (51, 56). We therefore hypothesized that thenitrite reductase activity of Ngb may regulate the hypoxic inhi-bition of cellular respiration by NO binding to cytochrome coxidase. This pathway provides an alternative explanation ofthe protective action of neuroglobin.NO can inhibit mitochondrial respiration and activate solu-

ble guanylyl cyclase at picomolar concentrations. For this rea-son, it is possible that changes in the disulfide bond may pro-duce enough NO to sustain physiologically relevant NOsynthesis rates (Fig. 8).Consistent with this thesis, our data (Fig. 5) demonstrate an

interaction between nitrite and deoxygenated neuroglobin thatgenerates bioavailable NO. In Fig. 6A, using isolatedmitochon-dria in the presence of nitrite, we observe more effective inhi-bition of respiration with wild type Ngb than the C55Amutant.However, the H64L and H64Q mutants indicate that fast ratesof NO generation are within the reach of the enzyme. Theextent of mitochondrial inhibition is dependent on the hemecoordination structure of neuroglobin and intrinsic nitritereductase activity.We speculate that the observed redox effectsmay be amplified by external effects such as changes in pH andprotein-protein modifications, namely protein phosphoryla-tion and/or protein-protein interactions, that further open upthe enzyme under allosteric control.In conclusion, the molecular examination of critical heme

pocket and surface thiol amino acids, using site-directedmutagenesis, provides a novel understanding of neuroglobinfunctionality as an enzyme with a redox-regulated six-to-five-coordinate iron heme transition that directs nitrite in the hemepocket for controlled electron and proton transfer reactions toform NO. The results presented in this study support the pro-vocative hypothesis that the cellular six-coordinate heme glo-bins, neuroglobin, cytoglobin, D. melanogaster hemoglobin,and plant hemoglobins, may subserve a function as primordialallosterically redox-regulatedNO-signaling proteins. The iden-tification of other allosteric regulators of the six-to-five coordi-nation of the neuroglobin heme pocket may reveal new intra-cellular mechanisms for controlling NO signaling via nitritereduction.

Acknowledgments—We thank Dr. Li Yi for helpful advice and discus-sions on disulfide couple redox potential determination and Dr.Yinna Wang for excellent technical assistance. Sperm whale myoglo-bins were a gift from John S. Olson (Rice University).

REFERENCES1. Hankeln, T., Ebner, B., Fuchs, C., Gerlach, F., Haberkamp,M., Laufs, T. L.,

Roesner, A., Schmidt, M., Weich, B., Wystub, S., Saaler-Reinhardt, S.,Reuss, S., Bolognesi, M., De Sanctis, D., Marden, M. C., Kiger, L., Moens,L., Dewilde, S., Nevo, E., Avivi, A., Weber, R. E., Fago, A., and Burmester,T. (2005) J. Inorg. Biochem. 99, 110–119

2. Brunori, M., and Vallone, B. (2007) Cell. Mol. Life Sci. 64, 1259–12683. Burmester, T., Haberkamp, M., Mitz, S., Roesner, A., Schmidt, M., Ebner,

B., Gerlach, F., Fuchs, C., andHankeln, T. (2004) IUBMB Life 56, 703–7074. Dewilde, S., Kiger, L., Burmester, T., Hankeln, T., Baudin-Creuza, V.,

Aerts, T.,Marden,M. C., Caubergs, R., andMoens, L. (2001) J. Biol. Chem.276, 38949–38955

5. Uzan, J., Dewilde, S., Burmester, T., Hankeln, T., Moens, L., Hamdane, D.,Marden, M. C., and Kiger, L. (2004) Biophys. J. 87, 1196–1204

6. Capece, L., Marti, M. A., Bidon-Chanal, A., Nadra, A., Luque, F. J., andEstrin, D. A. (2009) Proteins 75, 885–894

7. Kriegl, J. M., Bhattacharyya, A. J., Nienhaus, K., Deng, P., Minkow, O., andNienhaus, G. U. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 7992–7997

8. Kiger, L., Uzan, J., Dewilde, S., Burmester, T., Hankeln, T., Moens, L.,Hamdane, D., Baudin-Creuza, V., andMarden, M. (2004) IUBMB Life 56,709–719

9. Giuffre, A., Moschetti, T., Vallone, B., and Brunori, M. (2008) Biochem.Biophys. Res. Commun. 367, 893–898

10. Burmester, T., and Hankeln, T. (2009) J. Exp. Biol. 212, 1423–142811. Greenberg, D. A., Jin, K., and Khan, A. A. (2008)Curr. Opin. Pharmacol. 8,

20–2412. Khan, A. A., Wang, Y., Sun, Y., Mao, X. O., Xie, L., Miles, E., Graboski, J.,

Chen, S., Ellerby, L. M., Jin, K., and Greenberg, D. A. (2006) Proc. Natl.Acad. Sci. U.S.A. 103, 17944–17948

13. Wang, X., Liu, J., Zhu, H., Tejima, E., Tsuji, K., Murata, Y., Atochin, D. N.,Huang, P. L., Zhang, C., and Lo, E. H. (2008) Stroke 39, 1869–1874

14. Sun, Y., Jin, K.,Mao, X.O., Zhu, Y., andGreenberg, D. A. (2001) Proc. Natl.Acad. Sci. U.S.A. 98, 15306–15311

15. Weiland, T. R., Kundu, S., Trent, J. T., 3rd, Hoy, J. A., and Hargrove, M. S.(2004) J. Am. Chem. Soc. 126, 11930–11935

16. Nadra, A. D., Martí, M. A., Pesce, A., Bolognesi, M., and Estrin, D. A.(2008) Proteins 71, 695–705

17. Garrocho-Villegas, V., Gopalasubramaniam, S. K., and Arredondo-Peter,R. (2007) Gene 398, 78–85

18. Huang, Z., Shiva, S., Kim-Shapiro, D. B., Patel, R. P., Ringwood, L. A., Irby,C. E., Huang, K. T., Ho, C., Hogg, N., Schechter, A. N., and Gladwin,M. T.(2005) J. Clin. Invest. 115, 2099–2107

19. Shiva, S., Huang, Z., Grubina, R., Sun, J., Ringwood, L. A., MacArthur,P. H., Xu, X.,Murphy, E., Darley-Usmar, V.M., andGladwin,M. T. (2007)Circ. Res. 100, 654–661

20. Hendgen-Cotta, U. B.,Merx,M.W., Shiva, S., Schmitz, J., Becher, S., Klare,J. P., Steinhoff, H. J., Goedecke, A., Schrader, J., Gladwin, M. T., Kelm, M.,and Rassaf, T. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 10256–10261

21. Basu, S., Azarova, N. A., Font, M. D., King, S. B., Hogg, N., Gladwin,M. T.,Shiva, S., and Kim-Shapiro, D. B. (2008) J. Biol. Chem. 283, 32590–32597

22. Hamdane, D., Kiger, L., Dewilde, S., Green, B. N., Pesce, A., Uzan, J.,Burmester, T., Hankeln, T., Bolognesi, M., Moens, L., and Marden, M. C.(2003) J. Biol. Chem. 278, 51713–51721

23. Hamdane, D., Kiger, L., Dewilde, S., Green, B. N., Pesce, A., Uzan, J.,Burmester, T., Hankeln, T., Bolognesi, M., Moens, L., and Marden, M. C.(2004)Micron 35, 59–62

24. Nicolis, S., Monzani, E., Ciaccio, C., Ascenzi, P., Moens, L., and Casella, L.(2007) Biochem. J. 407, 89–99

25. Grubina, R., Huang, Z., Shiva, S., Joshi, M. S., Azarov, I., Basu, S., Ring-wood, L. A., Jiang, A., Hogg, N., Kim-Shapiro, D. B., and Gladwin, M. T.(2007) J. Biol. Chem. 282, 12916–12927

26. Burmester, T., Weich, B., Reinhardt, S., and Hankeln, T. (2000) Nature407, 520–523

27. Grassetti, D. R., and Murray, J. F., Jr. (1967) Arch. Biochem. Biophys. 119,41–49

28. Pesce, A., Dewilde, S., Nardini, M., Moens, L., Ascenzi, P., Hankeln, T.,Burmester, T., and Bolognesi, M. (2003) Structure 11, 1087–1095




http://ww

w.jbc.org/

Dow

nloaded from

http://www.jbc.org/

29. Schwede, T., Kopp, J., Guex, N., and Peitsch, M. C. (2003) Nucleic AcidsRes. 31, 3381–3385

30. Yi, L., Jenkins, P. M., Leichert, L. I., Jakob, U., Martens, J. R., and Ragsdale,S. W. (2009) J. Biol. Chem. 284, 20556–20561

31. Azarov, I., Huang, K. T., Basu, S., Gladwin, M. T., Hogg, N., and Kim-Shapiro, D. B. (2005) J. Biol. Chem. 280, 39024–39032

32. Van Doorslaer, S., Dewilde, S., Kiger, L., Nistor, S. V., Goovaerts, E.,Marden, M. C., and Moens, L. (2003) J. Biol. Chem. 278, 4919–4925

33. Petersen, M. G., Dewilde, S., and Fago, A. (2008) J. Inorg. Biochem. 102,1777–1782

34. Grubina, R., Basu, S., Tiso, M., Kim-Shapiro, D. B., and Gladwin, M. T.(2008) J. Biol. Chem. 283, 3628–3638

35. Salhany, J. M. (2008) Biochemistry 47, 6059–607236. Wakasugi, K., Nakano, T., and Morishima, I. (2003) J. Biol. Chem. 278,

36505–3651237. Schafer, F. Q., and Buettner, G. R. (2001) Free Radic. Biol. Med. 30,

1191–121238. Du,W., Syvitski, R., Dewilde, S.,Moens, L., and LaMar, G.N. (2003) J. Am.

Chem. Soc. 125, 8080–808139. Xu, J., Li, L., Yin, G., Li, H., and Du, W. (2009) J. Inorg. Biochem. 103,

1693–170140. Crawford, J. H., Isbell, T. S., Huang, Z., Shiva, S., Chacko, B. K., Schechter,

A. N., Darley-Usmar, V. M., Kerby, J. D., Lang, J. D., Jr., Kraus, D., Ho, C.,Gladwin, M. T., and Patel, R. P. (2006) Blood 107, 566–574

41. Nienhaus, K., Kriegl, J. M., and Nienhaus, G. U. (2004) J. Biol. Chem. 279,22944–22952

42. Ikeda-Saito, M., Hori, H., Andersson, L. A., Prince, R. C., Pickering, I. J.,George, G.N., Sanders, C. R., 2nd, Lutz, R. S.,McKelvey, E. J., andMattera,R. (1992) J. Biol. Chem. 267, 22843–22852

43. Biram, D., Garratt, C. J., and Hester, R. E. (1993) Biochim. Biophys. Acta1163, 67–74

44. Gladwin, M. T., Grubina, R., and Doyle, M. P. (2009) Acc. Chem. Res. 42,

157–16745. Yi, J., Safo, M. K., and Richter-Addo, G. B. (2008) Biochemistry 47,

8247–824946. Basu, S., Grubina, R.,Huang, J., Conradie, J., Huang, Z., Jeffers, A., Jiang,A.,

He, X., Azarov, I., Seibert, R., Mehta, A., Patel, R., King, S. B., Hogg, N.,Ghosh, A., Gladwin,M. T., andKim-Shapiro, D. B. (2007)Nat. Chem. Biol.3, 785–794

47. Copeland, D.M., Soares, A. S.,West, A.H., and Richter-Addo,G. B. (2006)J. Inorg. Biochem. 100, 1413–1425

48. Hoshino,M.,Maeda,M., Konishi, R., Seki, H., and Ford, P. C. (1996) J. Am.Chem. Soc. 118, 5702–5707

49. Brancaccio, A., Cutruzzola, F., Allocatelli, C. T., Brunori, M., Smerdon,S. J., Wilkinson, A. J., Dou, Y., Keenan, D., Ikeda-Saito, M., and Brantley,R. E., Jr. (1994) J. Biol. Chem. 269, 13843–13853

50. Doyle, M. P., Pickering, R. A., DeWeert, T. M., Hoekstra, J. W., and Pater,D. (1981) J. Biol. Chem. 256, 12393–12398

51. Jung, K. H., Chu, K., Lee, S. T., Sunwoo, J. S., Park, D. K., Kim, J. H., Kim, S.,Lee, S. K., Kim, M., and Roh, J. K. (2010) Biochem. Biophys. Res. Commun.403, 66–72

52. Raychaudhuri, S., Skommer, J., Henty, K., Birch, N., and Brittain, T. (2010)Apoptosis 15, 401–411

53. Liu, J., Yu, Z., Guo, S., Lee, S. R., Xing, C., Zhang, C., Gao, Y., Nicholls,D. G., Lo, E. H., and Wang, X. (2009) J. Neurosci. Res. 87, 164–170

54. Brunori, M., Giuffre, A., Forte, E., Mastronicola, D., Barone, M. C., andSarti, P. (2004) Biochim. Biophys. Acta 1655, 365–371

55. Mason, M. G., Nicholls, P., Wilson, M. T., and Cooper, C. E. (2006) Proc.Natl. Acad. Sci. U.S.A. 103, 708–713

56. Presley, T. D., Morgan, A. R., Bechtold, E., Clodfelter, W., Dove, R. W.,Jennings, J. M., Kraft, R. A., King, S. B., Laurienti, P. J., Rejeski, W. J.,Burdette, J. H., Kim-Shapiro, D. B., and Miller, G. D. (2011) Nitric Oxide24, 34–42




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Daniel B. Kim-Shapiro and Mark T. GladwinSheila Frizzell, Thottala Jayaraman, Lisa Geary, Calli Shapiro, Chien Ho, Sruti Shiva,

Mauro Tiso, Jesús Tejero, Swati Basu, Ivan Azarov, Xunde Wang, Virgil Simplaceanu,Human Neuroglobin Functions as a Redox-regulated Nitrite Reductase

doi: 10.1074/jbc.M110.159541 originally published online February 4, 20112011, 286:18277-18289.J. Biol. Chem.

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