Peroxynitrite Detoxification by Human Haptoglobin:Hemoglobin

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Peroxynitrite Detoxification by Human Haptoglobin:Hemoglobin Complexes: a Comparative Study. Paolo Ascenzi, and Massimiliano Coletta J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b05340 • Publication Date (Web): 24 Jul 2018 Downloaded from http://pubs.acs.org on July 31, 2018

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The Journal of Physical Chemistry

Peroxynitrite Detoxification by Human Haptoglobin:Hemoglobin Complexes: a Comparative Study.†

Paolo Ascenzi a and Massimo Coletta b,c,*

a

Interdepartmental Laboratory for Electron Microscopy, Roma Tre University, Via della Vasca Navale 79, I-00146 Roma, Italy

b

Department of Clinical Sciences and Translational Medicine, University of Roma “Tor Vergata”, Via Montpellier 1, I-00133 Roma, Italy c

Interuniversity Consortium for the Research on the Chemistry of Metals in Biological Systems, Via Celso Ulpiani 27, I-70126 Bari, Italy

Running title: Peroxynitrite scavenging by ferric human haptoglobin:hemoglobin complexes

Abbreviations: Hb, human hemoglobin; Hb(II)-O2, ferrous oxygenated Hb; Hb(III), ferric Hb; Hp, human haptoglobin; Hp1-1, phenotype 1-1 of Hp; Hp2-2, phenotype 2-2 of Hp; Hp1-1:Hb, Hp11:Hb complex; Hp1-1:Hb(III), ferric Hp1-1:Hb(III) complex; Hp2-2:Hb, Hp2-2:Hb complex; Hp22:Hb(III), ferric Hp2-2:Hb(III) complex; SA, human serum albumin; SA-heme, heme-bound human serum albumin.

†

This paper is dedicated to W.A. Eaton on the occasion of his 80th anniversary.

*Corresponding author: Prof. Massimo Coletta, Department of Clinical Sciences and Translational Medicine, University of Roma Tor Vergata, Via Montpellier 1, I-00133 Roma, Italy Phone: +39-06-72596365; fax: +39-06-72596353; email address: [email protected]

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Abstract Haptoglobin (Hp) reacts with dimeric hemoglobin (Hb), shifting the equilibrium in favor of the αβ dimer, displaying heme-based catalysis. Here, kinetics of peroxynitrite scavenging by ferric human haptoglobin1-1: and haptoglobin2-2:hemoglobin complexes (Hp1-1:Hb(III) and Hp22:Hb(III), respectively) is reported between pH 6.2 and 8.3 at 20.0 °C. The reactivity of Hp11:Hb(III) and Hp2-2:Hb(III) against peroxynitrite is similar to that of tetrameric Hb(III), reflecting the R-like structure of the αβ dimers of Hb(III) bound to Hp. To investigate the protective role of Hp1-1:Hb(III) and Hp2-2:Hb(III) against peroxynitrite-mediated nitration, the relative yield of nitro-L-tyrosine formed by the reaction of peroxynitrite with free

L-tyrosine

was determined.

Interestingly, both Hp1-1:Hb(III) and Hp2-2:Hb(III) impair peroxynitrite-mediated nitration of free L-tyrosine.

Therefore, Hp:Hb complexes could participate to the detoxification of reactive nitrogen

and oxygen species in vivo, contributing to prevent extra-erythrocytic Hb-induced damage during hemolytic crisis.


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Introduction Hemoglobin (Hb), the most prominent intracellular protein in blood, carries O2 in the circulatory system and participates to the metabolism of reactive oxygen and nitrogen species.1-7 The release of Hb and free heme into plasma is a physiological phenomenon that occurs during the hemolysis of senescent erythrocytes and the enucleation of erythroblasts. Moreover, massive intravascular hemolysis is a severe pathological complication of autoimmune, infectious, and inherited disorders, such as in sickle cell disease.1,8 This event can become of great relevance in the case of sickle cell disease, since one of therapeutic approaches being presently developed is the swelling of the erythrocytes,9 which might bring about a relevant hemolytic crisis as a countereffect. Free heme, arising from the degradation of Hb, is trapped by high and low density lipoproteins, serum albumin (SA), and hemopexin, thereby ensuring its complete clearance. Then, hemopexin releases the heme into hepatic parenchymal cells only after internalization of the hemopexin-heme complex by CD91 receptor-mediated endocytosis.8,10-12 On the other hand, αβ Hb dimers interact in the plasma with haptoglobin (Hp), forming complexes which are subsequently delivered to the reticulo-endothelial system by CD163 receptor-mediated endocytosis.8,11-15 The polymorphic nature of the Hp gene results in different levels of the antioxidant activity of the Hp:Hb complexes playing a immunomodulatory role in parasitic (malaria), bacterial (tuberculosis), viral (HIV) infections and non-infectious diseases (diabetic cardiovascular disease, obesity, retinopathy in type 2 diabetic patients, multiple sclerosis, and delayed cerebral ischemia).1622

In particular, the Hp2-2:Hb complex displays a decreased antioxidant capability, leading to

increased levels of oxidized lipids and decreased lipoprotein functions, and is cleared less efficiently from the circulation, promoting a buildup of iron in the plasma and in tissues (Goldenstein et al., 2012).19 Since Hp:Hb complexes, as well as heme-bound SA (SA-heme), high and low density lipoproteins, and hemopexin display transient heme-based ligand binding and (pseudo-)enzymatic properties,23-30 peroxynitritea detoxification by Hp1-1:Hb(III) and Hp23 ACS Paragon Plus Environment


2:Hb(III) complexes has been investigated here, between pH 6.2 and 8.3 at 20.0 °C. These data indicate that the reactivity of Hp1-1:Hb(III) and Hp2-2:Hb(III) against peroxynitrite is similar to that of tetrameric Hb(III), reflecting the R-like structure of the αβ dimers of Hb(III) bound to Hp. Therefore, Hp1-1:Hb(III) and Hp2-2:Hb(III) could participate to the detoxification of reactive nitrogen species and prevent extra-erythrocytic Hb-induced damage in vivo during hemolytic crisis.

Experimental Section Materials Human Hp1-1 and Hp2-2 were obtained from Athens Research & Technology, Inc. (Athens, GA, USA). Oxygenated human Hb (Hb(II)-O2) was prepared as previously reported.31 The Hp11:Hb(II)-O2 and Hp2-2:Hb(II)-O2 complexes were prepared by mixing Hb(II)-O2 with Hp1-1 and Hp2-2 at pH 7.0 and 20.0 °C, according to literature.32 The monomeric Hp:dimeric Hb(II)-O2 stoichiometry was 1:1. To avoid the presence of free Hb(II)-O2 a 20% excess of Hp1-1 and Hp2-2 was present in all samples, the absence of free Hb was checked by gel electrophoresis.32 Hp11:Hb(III) and Hp2-2:Hb(III) were obtained by adding few grains of ferricyanide to the protein solutions. Then, the excess of ferrycianide and by-products were removed by passing the solution through a Sephadex® G10 column (Sigma-Aldrich, St. Louis, MO, USA). Peroxynitrite was purchased from Cayman Chemical (Ann Arbor, Michigan, USA). The concentration of peroxynitrite was determined spectrophotometrically prior to each experiment by measuring the absorbance at 302 nm (ε302nm = 1.705×103 M−1 cm−1).33 L-Tyrosine L-Tyrosine

and nitro-L-tyrosine were obtained from Sigma-Aldrich (St. Louis, MO, USA).

and nitro-L-tyrosine were dissolved in 5.0×10–2 M phosphate buffer, at pH 7.0 and 20.0

°C; the final L-tyrosine concentration was 1.0×10–4 M.34-37


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All the other chemicals were purchased from Merck AG (Darmstadt, Germany) and SigmaAldrich (St. Louis, MO, USA). All chemicals were of analytical grade and were used without further puriﬁcation.

Methods Peroxynitrite isomerization by Hp1-1:Hb(III) and Hp2-2:Hb(III) was carried out between pH 6.2 and 8.3, at 20.0 °C, after degassing and keeping under nitrogen the protein solution.34-37 The effect of sodium azide on peroxynitrite isomerization by Hp1-1:Hb(III) and Hp22:Hb(III) was investigated by adding saturating levels of sodium azide (final concentration, 1.0×10−2 M) to the Hp1-1:Hb(III) and Hp2-2:Hb(III) solutions. This sodium azide concentration allows more than 95% formation of Hp1-1:Hb(III)- and Hp2-2:Hb(III)-azide complexes, as from the measured values of the dissociation equilibrium constant for sodium azide binding to Hp11:Hb(III) and Hp2-2:Hb(III) (i.e., Kd = 2.5(±0.3)×10-4 M and 3.1(±0.4)×10-4 M, respectively). The NO2– and NO3– levels were determined spectrophotometrically at 543 nm by using the Griess reagent and VCl3 to catalyze the conversion of NO3– to NO2–. The samples were prepared by mixing 0.5 mL of Hp1-1:Hb(III) and Hp2-2:Hb(III) (final concentration, 3.0×10–5 M in 5.0×10–2 M phosphate buffer, pH 7.0) with 0.5 mL of a peroxynitrite solution (final concentration, 2.0×10–4 M in 1.0×10–2 M NaOH) while vortexing, at 20.0 °C, in the absence and presence of sodium azide (final concentration, 1.0×10–2 M). The reaction mixture was analyzed within 10 min.34,36-38 The reaction of peroxynitrite with free L-tyrosine was carried out at pH 7.0 and 20.0 °C by adding 0.2 mL of an alkaline (1.0×10–2 M NaOH) ice-cooled solution of peroxynitrite (2.0×10–3 M) to 1.8 ml of a buffered (5.0×10−2 M phosphate buffer) solution of L-tyrosine (final concentration, 1.0×10−4 M) in the absence and presence of Hp1-1:Hb(III), Hp2-2:Hb(III), Hp1-1:Hb(III)-azide, and Hp2-2:Hb(III)-azide (the final concentration of Hp1-1:Hb(III), Hp2-2:Hb(III), Hp1-1:Hb(III)azide, and Hp2-2:Hb(III)-azide was 3.0×10–5 M; the final concentration of azide was 1.0×10–2 M). The amount of nitro-L-tyrosine was determined by HPLC analysis.34-37 5 ACS Paragon Plus Environment


Kinetics of peroxynitrite isomerization was investigated by rapid mixing the Hp1-1:Hb(III) or Hp2-2:Hb(III) solution (final concentration ranging between 5.0×10–6 M and 3.0×10–5 M) with the peroxynitrite solution (final concentration ranging between 3.0×10–5 M and 2.0×10–4 M), between pH 6.2 and 8.3 (5.0×10−2 M phosphate buffer), at 20.0 °C, in the absence and presence of sodium azide.34-37 Kinetics of peroxynitrite isomerization was recorded by using the SMF-2000 rapid-mixing stopped-flow apparatus (Bio-Logic SAS, Claix, France) monitoring absorbance changes at 302 nm, the characteristic absorbance spectroscopic maximum of peroxynitrite,33,39 and between 370 and 460 nm, the characteristic absorbance spectroscopic region of Hp1-1:Hb(III) and Hp2-2:Hb(III);40 the light path of the observation chamber was 10 mm, and the dead-time was 1.3 ms. Of note, only the absorbance decrease at 302 nm, reflecting peroxynitrite isomerization,33,39 was observed. The reaction of ferric heme-proteins (e.g., Hp:Hb(III) complexes) and heme-model compounds with peroxynitrite, in the absence and presence of sodium azide or cyanide (i.e., L), leads to the formation of the transient heme-Fe(III)-OONO species, which releases vary rapidly NO3− (see Scheme 1).33-37,39,41-50

Scheme 1. Mechanism of peroxynitrite isomerization by ferric heme-proteins and heme-model compounds (i.e., heme-Fe(III)).

In the absence and presence of sodium azide, values of the pseudo-first-order rate constant for peroxynitrite isomerization by Hp1-1:Hb(III) and Hp1-1:Hb(III) (i.e., kobs) were determined from the analysis of the time-dependent absorbance decrease at 302 nm, according to Eq. 1:34-37,44-50 6 ACS Paragon Plus Environment

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[peroxynitrite]t = [peroxynitrite]i × e – kobs × t

(1)

Values of the second-order rate constant for peroxynitrite isomerization by Hp1-1:Hb(III) and Hp1-1:Hb(III) (i.e., kon) and of the first-order rate constant for the spontaneous decay of peroxynitrite (i.e., k0) were obtained according to Eq. 2:34-37,44-50 kobs = kon × [peroxynitrite] + k0

(2)

The effect of pH on values of kon and k0 for peroxynitrite isomerization was analyzed according to Eq. 3:34-37,49,51,52 k = (klim × 10–pH) / (10–pH + 10–pKa)

(3)

where k indicates kon or k0, and klim represents the top asymptotic value of kon or k0 under conditions where pH 5.0×10–5 M,34 or the slow dissociation of the peroxynitrite/peroxynitrous acid adduct preceding the Hp1-1:Hb(III)- and Hp22:Hb(III)-mediated isomerization of peroxynitrite. As reported for heme-proteins and heme-model compounds,34,36,37,49,53 the isomerization of peroxynitrite in the absence of Hp1-1:Hb(III)- and Hp2-2:Hb(III), as well as in the presence of Hp11:Hb(III)-azide and Hp2-2:Hb(III)-azide, yields average values of 74±6% NO3− and 27±5% NO2−. However, the average values of NO3− and NO2− are 90±6% and 11±3%, respectively, in the presence of Hp1-1:Hb(III) and Hp2-2:Hb(III) (Table 2), clearly indicating that Hp:Hb complexes induce further production of NO3− (see Scheme 1).


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The effect of pH on values of k0 and kon for peroxynitrite isomerization was investigated to identify tentatively the species that preferentially react(s) with Hp1-1:Hb(III) and Hp2-2:Hb(III). Values of kon and k0 increase upon decreasing pH from 8.3 to 6.2 (Fig. 3). The pH dependence of kon values for the isomerization of peroxynitrite catalyzed from Hp1-1:Hb(III) and Hp2-2:Hb(III) (pKa = 6.8±0.3 and 6.8±0.2, respectively) is closely similar to that of k0 for peroxynitrite isomerization in the absence of Hp1-1:Hb(III) and Hp2-2:Hb(III) (pKa = 6.8±0.2). The pKa values for the pH dependence of kon and k0 here determined match well with pKa values reported in the literature,33,3537,41,49

indeed suggesting that HOONO is the species that undergoes preferentially the Hp1-

1:Hb(III)- and Hp2-2:Hb(III)-catalyzed and spontaneous isomerization of peroxynitrite (Scheme 1). To analyze the protective role of Hp1-1:Hb(III) and Hp2-2:Hb(III) against peroxynitritemediated nitration of free L-tyrosine, the relative yield of nitro-L-tyrosine formed by the reaction of peroxynitrite with free L-tyrosine in the absence and presence of Hp1-1:Hb(III), Hp2-2:Hb(III), Hp1-1:Hb(III)-azide, and Hp2-2:Hb(III)-azide was determined (Fig. 4). Hp1-1:Hb(III) and Hp22:Hb(III) protect in a dose-dependent fashion free nitration. In contrast,

L-tyrosine

L-tyrosine

against peroxynitrite-mediated

nitration is not prevented by Hp1-1:Hb(III)-azide, and Hp2-

2:Hb(III)-azide, the yield of nitro-L-tyrosine corresponding to that determined in the absence of Hp1-1:Hb(III) and Hp2-2:Hb(III).

Discussion Thanks to their plasmatic concentration, Hp (3 to 6×10−6 M)54 and SA (~7.5×10−4 M)29 play a relevant role in attenuating the damage stemming from the extra-erythrocytic presence of Hb and free heme, respectively.8,10-15,29 In turn, Hp:Hb complexes and SA-heme show heme-based ligand binding and (pseudo-)enzymatic properties28-31,55 and possibly play a role in vivo. Therefore, the comparison of peroxynitrite and/or NO scavenging by tetrameric Hb,28,34 Hp1-1:Hb and Hp2-2:Hb complexes (present study and refs28,31), SA-heme,29,36 and cardiolipin-bound cytochrome c (CL-cyt 9 ACS Paragon Plus Environment


c), present in the serum under pathological conditions,44,45,56-58 may be of relevance to estimate the contribution of these species to the clearance of toxic reactive nitrogen and oxygen species. It must be underlined that various reactive nitrogen and oxygen species undergo different detoxification reactions by hemoproteins according to their oxidation state, peroxynitrite being isomerized by ferric hemoproteins to NO3‒ while NO is oxidized to NO3‒ by oxygenated species; therefore, in the first case the hemoprotein works as a catalyst whereas for NO oxidation the hemoprotein acts a redox partner through the formation of an intermediate heme-Fe(III)-OONO‒ species (see Scheme 2). The inspection of Table 3, showing values of the second-order rate constant (kon) for peroxynitrite and/or NO scavenging in the absence and presence of heme-bound proteins, allows the following considerations. (i) Values of kon for Hp1-1:Hb(III)- and Hp2-2:Hb(III)-mediated isomerization of peroxynitrite are essentially similar (only about 25% faster) to that reported for peroxynitrite scavenging by tetrameric Hb(III).27 This result confirms that Hp:Hb(III) complexes and tetrameric Hb(III) possess a closely similar R-state structure.59-61 (ii) An even more efficient role for peroxynitrite isomerization can be played by SAheme(III), which, beside being much more concentrated, turns out to be at least ten-fold faster.36 (iii) Peroxynitrite isomerization by penta-coordinated CL-cytc,44 which is released by cells undergoing apoptosis,57 is very fast. However, the low concentration of CL-cytc in the serum58 renders its role in peroxynitrite detoxification much less relevant than that of Hp1-1:Hb, Hp2-2:Hb, Hb and SA-heme. (iv) In the case of CL-cytc, the role of the protein matrix seems very relevant, since the undecapeptide-bound heme c (i.e., MP-11) shows a much slower rate of peroxynitrite isomerization,49 which appears only slightly faster than that of Hp:Hb complexes. This point is of the utmost importance, since it casts light on how peroxynitrite detoxification is accomplished, requiring not only an easy access to the heme-Fe(III) atom, but, even more importantly, an efficient 10 ACS Paragon Plus Environment

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trapping of the peroxynitrite close to the metal center, which is effectively realized by slowing down the dissociation rate. As a whole, peroxynitrite capturing from solvent-exposed heme-Fe(III) (as occurs in MP11-Fe(III)) is much less efficient than from a metal center shielded from the solvent by the protein matrix. In particular, peroxynitrite is most efficiently trapped in SA-heme and CL-bound cytochrome c and somewhat less efficiently in tetrameric Hb(III) and Hp-bound αβ Hb(III) dimers. (v) NO scavenging by ferrous oxygenated tetrameric Hb(II)-O2,62-64 as well as Hp11:Hb(II)-O2 and Hp2-2:Hb(II)-O2,28 is characterized by the transient formation of the peroxynitritebound heme-Fe(II) complex preceding the release of NO3– (see Scheme 2).

kon

fast

heme-Fe(II)-O2 + NO → heme-Fe(III)-OONO− → heme-Fe(III) + NO3–

Scheme 2. Mechanism of NO scavenging by ferrous oxygenated heme-proteins (i.e., heme-Fe(II)-O2).

NO binding to the heme-Fe(II)-O2 center of tetrameric Hb, Hp1-1:Hb(II)-O2 and Hp22:Hb(II)-O2 is the rate-limiting step of catalysis since the heme-Fe(III)-OONO− species was never detected. Values of kon for NO scavenging from Hb(II)-O2, Hp1-1:Hb(II)-O2, and Hp2-2:Hb(II)-O2 are identical, this being in agreement with the fact that tetrameric Hb(II)-O2 and Hp:Hb(II)-O2 complexes are in the R-state.28,59 In conclusion, Hp impairs the oxidative damage stemming from extra-erythrocytic Hb by capturing it; in turn, Hp:Hb complexes may participate to the detoxification of reactive nitrogen and oxygen species28 during hemolytic crisis.



Acknowledgments The grant of Excellence Departments MIUR (Legge 232/2016, Articolo 1, Comma 314 337) is gratefully acknowledged.


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32. Brunori, M.; Alfsen, A.; Saggese, U.; Antonini, E.; Wyman, J. Studies on the oxidationreduction potentials of heme proteins. VII. Oxidation-reduction equilibrium of hemoglobin bound to haptoglobin. J. Biol. Chem. 1968, 243, 2950-2954. 33. Goldstein, S.; Merényi, G. The chemistry of peroxynitrite: implications for biological activity. Methods Enzymol. 2008, 436, 49-61. 34. Herold, S.; Shivashankar, K. Metmyoglobin and methemoglobin catalyze the isomerization of peroxynitrite to nitrate. Biochemistry 2003, 42, 14036-14046. 35. Herold, S.; Kalinga, S.; Matsui, T.; Watanabe, Y. Mechanistic studies of the isomerization of peroxynitrite to nitrate catalyzed by distal histidine metmyoglobin mutants. J. Am. Chem. Soc. 2004, 126, 6945-6955. 36. Ascenzi, P.; di Masi, A.; Coletta, M.; Ciaccio, C.; Fanali, G.; Nicoletti, F.P.; Smulevich, G.; Fasano, M. Ibuprofen impairs allosterically peroxynitrite isomerization by ferric human serum heme-albumin. J. Biol. Chem. 2009, 284, 31006-31017. 37. Ascenzi, P.; Pesce, A. Peroxynitrite scavenging by Campylobacter jejuni truncated hemoglobin P. J. Biol. Inorg. Chem. 2017, 22, 1141-1150. 38. Miranda, K.M.; Espey, M.G.; Wink, D.A. A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide 2001, 5, 62-71. 39. Goldstein, S.; Lind, J.; Merényi, G. Chemistry of peroxynitrites and peroxynitrates. Chem. Rev. 2005, 105, 2457-2470. 40. Mollan, T.L.; Jia, Y.; Banerjee, S.; Wu, G.; Kreulen, R.T.; Tsai, A.L.; Olson, J.S.; Crumbliss, A.L.; Alayash, A.I. Redox properties of human hemoglobin in complex with fractionated dimeric and polymeric human haptoglobin. Free Radic. Biol. Med. 2014, 69, 265-277. 41. Mehl, M.; Daiber, A.; Herold, S.; Shoun, H.; Ullrich, V. Peroxynitrite reaction with heme proteins. Nitric Oxide 1999, 3, 142-152. 16 ACS Paragon Plus Environment

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42. Shimanovich, R.; Groves, J.T. Mechanisms of peroxynitrite decomposition catalyzed by FeTMPS, a bioactive sulfonated iron porphyrin. Arch. Biochem. Biophys. 2001, 387, 307317. 43. Jensen, M.P.; Riley, D.P. Peroxynitrite decomposition activity of iron porphyrin complexes. Inorg. Chem. 2002, 41, 4788-4797. 44.Ascenzi, P.; Ciaccio, C.; Sinibaldi, F.; Santucci, R.; Coletta, M. Cardiolipin modulates

allosterically peroxynitrite detoxification by horse heart cytochrome c. Biochem. Biophys. Res. Commun. 2011, 404, 190-194. 45. Ascenzi, P.; Ciaccio, C.; Sinibaldi, F.; Santucci, R.; Coletta, M. Peroxynitrite detoxification by horse heart carboxymethylated cytochrome c is allosterically modulated by cardiolipin. Biochem. Biophys. Res. Commun. 2011, 415, 463-467. 46. Ascenzi, P.; Coletta, A.; Cao, Y.; Trezza, V.; Leboffe, L.; Fanali, G.; Fasano, M.; Pesce, A.; Ciaccio, C.; Marini, S.; Coletta, M. Isoniazid inhibits the heme-based reactivity of Mycobacterium tuberculosis truncated hemoglobin N. PLoS One 2013, 8, e69762. 47. Coppola, D.; Giordano, D.; Tinajero-Trejo, M.; di Prisco, G.; Ascenzi, P.; Poole, R.K.; Verde, C. Antarctic bacterial haemoglobin and its role in the protection against nitrogen reactive species. Biochim. Biophys. Acta 2013, 1834, 1923-1931. 48. Ascenzi, P.; Leboffe, L.; Pesce, A.; Ciaccio, C.; Sbardella, D.; Bolognesi, M.; Coletta, M. Nitrite-reductase and peroxynitrite isomerization activities of Methanosarcina acetivorans protoglobin. PLoS One 2014, 9, e95391. 49. Ascenzi, P.; Leboffe, L.; Santucci, R.; Coletta, M. Ferric microperoxidase-11 catalyzes peroxynitrite isomerization. J. Inorg. Biochem. 2015, 144, 56-61. 50. Coppola, D.; Giordano, D.; Milazzo, L.; Howes, B.D.; Ascenzi, P.; di Prisco, G.; Smulevich, G.; Poole, R.K.; Verde, C. Coexistence of multiple globin genes conferring protection against nitrosative stress to the Antarctic bacterium Pseudoalteromonas haloplanktis TAC125. Nitric Oxide 2018, 73, 39-51. 17 ACS Paragon Plus Environment


51. Pfeiffer, S.; Gorren, A.C.; Schmidt, K.; Werner, E.R.; Hansert, B.; Bohle, D.S.; Mayer, B. Metabolic fate of peroxynitrite in aqueous solution: reaction with nitric oxide and pHdependent decomposition to nitrite and oxygen in a 2:1 stoichiometry. J. Biol. Chem. 1997, 272, 3465-3470. 52. Herold, S.; Matsui, T.; Watanabe, Y. Peroxynitrite isomerization catalyzed by His64 myoglobin mutants. J. Am. Chem. Soc. 2001, 123, 4085-4086. 53. Kissner, R.; Koppenol, W.H. Product distribution of peroxynitrite decay as a function of pH, temperature, and concentration. J. Am. Chem. Soc. 2002, 124, 234-239. 54. Bacq, Y.; Schillio, Y.; Brechot, J.F.; De Muret, A.; Dubois, F.; Metman, E.H. Decrease of haptoglobin serum level in patients with chronic viral hepatitis C. Gastroenterol. Clin. Biol. 1993, 17, 364-369. 55. Ascenzi, P.; di Masi, A.; Fanali, G.; Fasano, M. Heme-based catalytic properties of human serum albumin. Cell Death Discov. 2015, 1, 15025. 56. Li, P.; Nijhawan, D.; Budihardjo, I.; Srinivasula, S.M.; Ahmad, M.; Alnemri, E.S.; Wang, X. Cytochrome c and d-ATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 1997, 91, 479-489. 57. Caroppi, P.; Sinibaldi, F.; Fiorucci, L.; Santucci, R. Apoptosis and human disease: mitochondrion damage and lethal role of cytochrome c as a proapoptotic protein. Curr. Med. Chem. 2009, 16, 4058-4065. 58. Hüttermann, M.; Pecina, P.; Rainbolt, M.; Sanderson, T.H.; Kagan, V.E.; Samavati, L.; Doan, J.W.; Lee, I. The multiple functions of cytochrome c and their regulation in life and death decisions of the mammalian cell: from respiration to apoptosis. Mitochondrion 2011, 11, 369-381. 59. Perutz, M.F. Regulation of oxygen affinity of hemoglobin: influence of structure of the globin on the heme iron. Annu. Rev. Biochem. 1979, 48, 327-386.


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60. Andersen, C.B.; Torvund-Jensen, M.; Nielsen, M.J.; de Oliveira, C.L.; Hersleth, H.P.; Andersen, N.H.; Pedersen, J.S.; Andersen, G.R.; Moestrup, S.K. Structure of the haptoglobin-haemoglobin complex. Nature 2012, 489, 456-459. 61. Stødkilde, K.; Torvund-Jensen, M.; Moestrup, S.K.; Andersen, C.B. Structural basis for trypanosomal haem acquisition and susceptibility to the host innate immune system. Nat. Commun. 2014, 5, 5487. 62. Doyle, M.P.; Hoekstra, J.W. Oxidation of nitrogen-oxides by bound dioxygen in hemoproteins. J. Inorg. Biochem. 1981, 14, 351-358. 63. Eich, R.F.; Li, T.S.; Lemon, D.D.; Doherty, D.H.; Curry, S.R.; Aitken, J.F.; Mathews, A.J.; Johnson, K.A.; Smith, R.D.; Phillips, G.N.; Olson, J.S. Mechanism of NO-induced oxidation of myoglobin and hemoglobin. Biochemistry 1996, 35, 6976-6983. 64. Herold, S.; Exner, M.; Nauser, T. Kinetic and mechanistic studies of the NO center dotmediated oxidation of oxymyoglobin and oxyhemoglobin. Biochemistry 2001, 40, 33853395.



Footnotes Footnote to page 4 a

The recommended IUPAC nomenclature for peroxynitrite is oxoperoxonitrate, and the

recommended nomenclature for peroxynitrous acid is hydrogen oxoperoxonitrate.33,39 The term “peroxynitrite” is used here to refer generically to both ONOO− and its conjugate acid HOONO.


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Table 1. pH dependence of kon and k0 values for peroxynitrite isomerization by Hp1-1:Hb(III) and Hp2-2:Hb(III), at 20.0 °C. a -----------------------------------------------------------------------------------------------------------------------Hp1-1:Hb(III) Hp2-2:Hb(III) ------------------------------------------------------------------------------------------------------------pH kon (M−1 s−1) k0 (s−1) pH kon (M−1 s−1) k0 (s−1) -----------------------------------------------------------------------------------------------------------------------6.2 (3.2±0.3)×104 (6.3±0.6)×10−1 6.4 (2.8±0.3)×104 (5.8±0.6)×10−1 6.6 (2.1±0.2)×104 (4.8±0.5)×10−1 6.7 (1.9±0.2)×104 (4.3±0.4)×10−1 7.0 (1.9±0.2)×104 (3.2±0.3)×10−1 7.0 (1.6±0.2)×104 (3.1±0.3)×10−1 7.3 (7.5±0.8)×103 (2.2±0.3)×10−1 7.4 (7.3±0.7)×103 (2.1±0.3)×10−1 3 −1 3 7.7 (4.1±0.4)×10 (1.3±0.2)×10 7.8 (3.6±0.3)×10 (1.1±0.2)×10−1 8.1 (2.1±0.2)×103 (8.1±0.9)×10−2 8.3 (1.2±0.1)×103 (7.1±0.8)×10−2 -----------------------------------------------------------------------------------------------------------------------a 5.0×10−2 M phosphate buffer.



Table 2. Nitrogen-containing products of peroxynitrite isomerization by Hp1-1:Hb(III) and Hp22:Hb(III), at pH 7.0 and 20.0 °C. a -----------------------------------------------------------------------------------------------------------------------Hp1-1:Hb(III) (M) [Azide] (M) NO3−(%) NO2− (%) -----------------------------------------------------------------------------------------------------------------------0.0 0.0 74±5 27±3 −5 3.0×10 0.0 91±6 9±2 −5 3.0×10 0.0 88±6 11±3 −5 −2 3.0×10 1.0×10 71±5 29±4 3.0×10−5 1.0×10−2 76±4 25±4 ======================================================================= Hp2-2:Hb(III) (M) [Azide] (M) NO3−(%) NO2− (%) -----------------------------------------------------------------------------------------------------------------------0.0 0.0 76±6 24±4 −5 3.0×10 0.0 88±5 12±3 −5 3.0×10 0.0 92±5 10±2 3.0×10−5 1.0×10−2 73±4 26±3 3.0×10−5 1.0×10−2 72±5 28±5 -----------------------------------------------------------------------------------------------------------------------a 5.0×10−2 M phosphate buffer.


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Table 3. Effect of human Hp1-1 and Hp2-2 on human Hb reactivity. -----------------------------------------------------------------------------------------------------------------------Reaction Heme-protein kon (M−1 s−1) -----------------------------------------------------------------------------------------------------------------------Peroxynitrite isomerization Hb(III) a 1.2×104 Hp1-1:Hb(III) b 1.7×104 Hp2-2:Hb(III) b 1.6×104 HSA-heme(III) c 4.1×105 d CL-cytc(III) 3.2×105 MP-11(III) e 4.1×104 NO scavenging f

Hb(II)-O2 6.0×106 Hp1-1:Hb(II)-O2 6.1×106 Hp2-2:Hb(II)-O2 6.0×106 ------------------------------------------------------------------------------------------------------------------------



Footnote to Table 3 a

pH 7.0 and 20.0 °C.34 b pH 7.0 and 20.0 °C. Present study. c pH 7.2 and 22 °C.36 d pH 7.0 and 20.0 °C.44 ([CL] = 1.6×10−4 M). e pH 7.2 and 20.0 °C.49 f pH and temperature are not mentioned.28


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Figure captions

Fig. 1. Kinetics of Hp1-1:Hb(III)- and Hp2-2:Hb(III)-mediated peroxynitrite isomerization, at 20.0 °C. (A) Averaged time courses of Hp1-1:Hb(III)-mediated isomerization of peroxynitrite, at pH 7.0. The time course analysis according to Eq. 1 allowed to determine the following values of kobs: trace a, kobs = 3.8×10−1 s−1; and trace b, kobs = 8.9×10−1 s−1. The Hp1-1:Hb(III) concentration was: trace a, 5.0×10−6 M; and trace b, 3.0×10−5 M. (B) Averaged time courses of Hp2-2:Hb(III)-mediated isomerization of peroxynitrite, at pH 7.0. The time course analysis according to Eq. 1 allowed to determine the following values of kobs: trace a, kobs = 3.6×10−1 s−1; and trace b, kobs = 8.1×10−1 s−1. The Hp1-1:Hb(III) concentration was: trace a, 5.0×10−6 M; and trace b, 3.0×10−5 M. (C) Dependence of kobs on the Hp1-1:Hb(III) concentration. Data obtained at pH 6.2, 7.0, and 7.7 were analyzed according to Eq. 2 with values of kon and k0 given in Table 1. (D) Dependence of kobs on the Hp2-2:Hb(III) concentration. Data obtained at pH 6.4, 7.0, and 7.8 were analyzed according to Eq. 2 with values of kon and k0 given in Table 1. (E) Dependence of kobs on the Hp1-1:Hb(III) concentration in the presence of azide, at pH 7.0. The symbol on the ordinate indicates the k0 value obtained in the absence of Hp1-1:Hb(III) ((3.2±0.3)×10–1 s–1); the k0 value corresponds to those of kobs determined in the presence of Hp1-1:Hb(III)-azide (the average value was (2.9±0.3)×10–1 s–1). (F) Dependence of kobs on the Hp2-2:Hb(III) concentration in the presence of azide, at pH 7.0. The symbol on the ordinate indicates the value of k0 obtained in the absence of Hp2-2:Hb(III) ((3.1±0.3)×10–1 s–1); the k0 value corresponds to those of kobs determined in the presence of Hp22:Hb(III)-azide (the average value was (3.2±0.3)×10–1 s–1). The peroxynitrite concentration was 2.0×10–4 M. The azide concentration was 1.0×10–2 M. Where not shown, the standard deviation is smaller than the symbol.



Fig. 2. Effect of peroxynitrite concentration on values of kobs for peroxynitrite isomerization from Hp1-1:Hb(III) (A) and Hp2-2:Hb(III) (B), at pH 7.0 and 20.0 °C. The Hp1-1:Hb(III) and Hp22:Hb(III) concentration was 5.0×10–6 M.

Fig. 3. Effect of pH on peroxynitrite isomerization, at 20.0 °C. (A) Effect of pH on values of kon for peroxynitrite isomerization from Hp1-1:Hb(III). The continuous line was calculated according to Eq. 3 with pKa = 6.8±0.3 and klim = (3.8±0.7)×104 M–1 s–1. (B) Effect of pH on values of kon for peroxynitrite isomerization from Hp2-2:Hb(III). The continuous line was calculated according to Eq. 3 with pKa = 6.8±0.2 and klim = (3.9±06)×104 M–1 s–1. (C) Effect of pH on values of k0 for peroxynitrite isomerization in the absence of Hp1-1:Hb(III) (filled circles) and Hp2-2:Hb(III) (filled squares). The continuous line was calculated according to Eq. 3 with pKa = 6.8±0.2 and klim = (8.0±0.7)×10–1 s–1. Where not shown, the standard deviation is smaller than the symbol.

Fig. 4. Effect of the concentration of Hp1-1:Hb(III) and Hp1-1:Hb(III)-azide (A; open and filled circles, respectively) and of Hp2-2:Hb(III) and Hp2-2:Hb(III)-azide (B; open and filled squares, respectively) on the relative yield of nitro-L-tyrosine formed from the reaction of peroxynitrite with free L-tyrosine, at pH 7.0 and 20.0 °C. The symbols on the ordinate indicate the relative yield of nitro-L-tyrosine obtained in the absence of Hp1-1:Hb(III), Hp1-1:Hb(III)-azide, Hp2-2:Hb(III), and Hp2-2:Hb(III)-azide. The free

L-tyrosine

concentration was 1.0×10–4 M. The peroxynitrite

concentration was 2.0×10–4 M. The azide concentration was 1.0×10–2 M. Relative nitro-L-tyrosine yield (%) = (yield with added Hp:Hb(III) / yield with no Hp:Hb(III)) × 100. Where not shown, the standard deviation is smaller than the symbol.


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A

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Figure 1

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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Figure 2

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[Peroxynitrite] × 10 (M)

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Figure 3

A k on × 10-4 (M-1 s-1)

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4

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Relative yield of nitro-L-tyrosine (%)

Figure 4

Relative yield of nitro-L-tyrosine (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

A 100 75 50 25 0 0

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Haptoglobin (Hp) binding to hemoglobin (Hb) is crucial to prevent extra-erythrocytic Hb-induced damage; in turn, Hp:Hb complexes display heme-based reactivity. Of note, ferrous human αβ Hb dimers complexed with the human Hp phenotypes 1-1 and 2-2 catalyze the conversion of peroxynitrite to NO3−. 77x69mm (300 x 300 DPI)

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Peroxynitrite Detoxification by Human Haptoglobin:Hemoglobin

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