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The heme-iron geometry of ferrous nitrosylated heme-serum lipoproteins, hemopexin, and albumin: a comparative EPR study
Journal of Inorganic Biochemistry 91 (2002) 487–490 www.elsevier.com / locate / jinorgbio
Short Communication
The heme-iron geometry of ferrous nitrosylated heme-serum lipoproteins, hemopexin, and albumin: a comparative EPR study a ,1
Mauro Fasano , Marco Mattu
b ,1
c b, , Massimo Coletta , Paolo Ascenzi *
a Department of Structural and Functional Biology, University of Insubria, Via Jean H. Dunant 3, I-21100 Varese, Italy Department of Biology and Interdepartmental Laboratory for Electron Microscopy, University ‘ Roma Tre’, Viale Guglielmo Marconi 446, I-00146 Rome, Italy c Department of Experimental Medicine and Biochemical Sciences, University of Rome ‘ Tor Vergata’, Via Montpellier 1, I-00133 Rome, Italy b
Received 5 March 2002; received in revised form 3 May 2002; accepted 24 May 2002 This study is dedicated to M.F. Perutz who pioneered studies on heme-proteins.
Abstract Serum high and low density lipoproteins, albumin, and hemopexin (HDL, LDL, SA, and HPX, respectively) serve as traps of toxic plasma heme and participate in its complete clearance by transportation to the liver. Moreover, SA-(heme) and HPX-heme have been proposed to facilitate NO scavenging in vivo. Here, the EPR-spectroscopic properties of ferrous nitrosylated heme-human high and low density lipoproteins (HDL-heme-NO and LDL-heme-NO, respectively) as well as of ferrous nitrosylated heme-rabbit serum hemopexin (HPX-heme-NO) are reported and analyzed in parallel with those of ferrous nitrosylated heme-human serum albumin (SA-heme-NO). HDL-heme-NO and LDL-heme-NO as well as SA-heme-NO, in the absence of allosteric effectors (i.e., N-form), are five-coordinate heme-iron species, characterized by the three-line splitting observed in the high magnetic field region of the X-band EPR spectrum. On the other hand, SA-heme-NO, in the presence of drugs (i.e., B-form), and HPX-heme-NO are six-coordinate heme-iron species, characterized by an X-band EPR spectrum with an axial geometry. The heme-iron coordination state of HDL-heme-NO, LDL-heme-NO, SA-heme-NO, and HPX-heme-NO is in keeping with values of ferric heme dissociation rate constants which decrease in the following order: LDL.HDL.SA.HPX. Altogether, these observations suggest that HPX displays a cleft much more suitable for heme binding than other heme-carriers. 2002 Elsevier Science Inc. All rights reserved. Keywords: Heme-human lipoproteins; Heme-rabbit hemopexin; Heme-human albumin; Ferrous nitrosylated heme-serum proteins; Heme-iron geometry; EPR spectroscopic properties
Plasma globin-free heme can stem from intracellular hemoglobin (Hb) degradation, which first leads to the accumulation of heme in red cell membrane followed by the heme dissociation from the membrane and binding to plasma proteins. Heme may also appear in plasma due to the heme release from extracellular Hb upon red cell lysis, under pathological conditions. Extracellular Hb tends to dissociate into dimers and the heme to oxidize to the ferric form which is then prone to be released from Hb to plasma (see Refs. [1–6]).
*Corresponding author. Tel.: 139-6-5517-6329; fax: 139-6-55176321. E-mail address: [email protected] (P. Ascenzi). 1 These authors contributed equally to this work.
Serum albumin, hemopexin as well as high and low density lipoproteins (SA, HPX, HDL, and LDL, respectively) serve as traps of the toxic plasma heme, thereby ensuring its complete clearance by transport to the liver (see Refs. [2–6]). Surprisingly, during the first seconds after heme appearance in plasma, more than 80% of this powerful oxidizer binds to HDL and LDL, the most oxidatively intolerant plasma components; and only the remaining 20% binds to antioxidative SA and HPX. SA and HPX slowly remove most of the heme from HDL and LDL (see Ref. [4]). Then, heme transits to HPX, that releases it into hepatic parenchymal cells only after internalization of HPX-heme by specific receptor-mediated endocytosis. After delivering the heme intracellularly, HPX is released intact into the bloodstream and the heme is degraded. Under conditions of severe hemolysis,
0162-0134 / 02 / $ – see front matter 2002 Elsevier Science Inc. All rights reserved. PII: S0162-0134( 02 )00473-7
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M. Fasano et al. / Journal of Inorganic Biochemistry 91 (2002) 487 – 490
hemopexin is overwhelmed and other processes come into play [3–6]. Binding of NO to SA-heme and HPX-heme suggested that these plasma heme-carriers may protect against NOmediated toxicity, especially in conditions of trauma and hemolysis. SA-heme and HPX-heme may facilitate NO scavenging by heme-iron chemistry. Moreover, SA-(heme) participates to NO metabolism by Cys34 (trans)-nitrosylation (see Refs. [2,7–9]). Here, the EPR-spectroscopic properties of ferrous nitrosylated heme-human high and low density lipoproteins as well as of ferrous nitrosylated heme-rabbit hemopexin (HDL-heme-NO, LDL-heme-NO, and HPX-heme-NO, respectively) are reported and analyzed in parallel with those of ferrous nitrosylated heme-human serum albumin (SA-heme-NO) (see Refs. [10,11]). Human serum HDL and LDL as well as heme chloride, NOR-1, and SNAP were obtained from Sigma (St. Louis, MO, USA). Rabbit serum HPX was prepared as previously reported (see Refs. [8,12]). Gaseous NO was purchased from Aldrich (Milwaukee, WI, USA). All the other products were purchased from Merck (Darmstadt, Germany). All chemicals were of analytical or reagent grade and used without further purification. HDL-heme (3.0310 25 M), LDL-heme (3.0310 25 M), and HPX-heme (2.8310 25 M) were prepared by adding 1.2-molar excess of HDL, LDL, and HPX to ferrous heme in 5.0310 22 M phosphate, pH 6.5 (HDL-heme and LDLheme) and 7.1 (HPX-heme); no free heme is present in solution (see Refs. [4,8,12]). The values of the dissociation equilibrium constant for heme binding to HDL, LDL, and HPX primary site(s) are lower than 10 29 M [4,8]. HDLheme-NO (3.0310 25 M), LDL-heme-NO (3.0310 25 M), and HPX-heme-NO (2.8310 25 M) were obtained, under anaerobic conditions, (i) by sequential addition of 10molar excess of sodium dithionite or sodium ascorbate and 4-molar excess of potassium nitrite to HDL-heme, LDLheme, and HPX-heme in 5.0310 22 M phosphate buffer, pH 6.5 or 7.1, (ii) by blowing purified NO over the HDL-heme, LDL-heme, and HPX-heme solution (5.03 10 22 M phosphate buffer, pH 6.5 or 7.1), in the absence and presence of 5-molar excess of sodium dithionite or sodium ascorbate, and (iii) by sequential addition of 10molar excess of dithiothreitol and 4-molar excess of NOR1 or SNAP to HDL-heme, LDL-heme, and HPX-heme in 5.0310 22 M phosphate buffer, pH 6.5 or 7.1 (see Refs. [7,8,10,11,13]). X-band EPR spectra of HDL-heme-NO, LDL-heme-NO, and HPX-heme-NO were collected at pH 6.5 or 7.1 (5.03 10 22 M phosphate buffer), and 110 K on a Bruker ESP 300 spectrometer with 100-kHz field modulation (see Refs. [10,11]). HDL-heme-NO, LDL-heme-NO, and HPX-hemeNO samples obtained by different methods gave identical X-band EPR spectra (data not shown). The X-band EPR spectra of HDL-heme-NO and LDLheme-NO as well as of SA-heme-NO, in the absence of
allosteric effectors (i.e., N-form) (see Fig. 1) display the same basic characteristics reported for five-coordinate ferrous nitrosylated heme-model compounds (e.g., a,b,g,dtetraphenylporphyrin (FeTTP-NO)) and heme-proteins (e.g., ferrous nitrosylated human adult hemoglobin (HbNO) obtained in the presence of allosteric effectors; i.e.,
Fig. 1. X-band EPR spectra of human HDL-heme-NO, human LDLheme-NO, human SA-heme-NO (in the absence of allosteric effectors and in the presence of drugs; i.e., N- and B-form, respectively), and rabbit HPX-heme-NO. X-band EPR spectra of SA-heme-NO were from Refs. [10,11]. X-band EPR spectra were obtained at pH 6.5 or 7.1, and 100 or 110 K. For further details, see text.
M. Fasano et al. / Journal of Inorganic Biochemistry 91 (2002) 487 – 490
T-state). Ferrous nitrosylated five-coordinate systems are characterized by the three-line splitting observed in the high magnetic field region of the X-band EPR spectrum (see Refs. [1,10,11,13–19]) (see Table 1). Interestingly, electronic absorption spectroscopy data support that SAheme-NO in the N-form is a five-coordinate heme-iron species [7]. The X-band EPR spectra of SA-heme-NO, in the presence of drugs (i.e., B-form), and HPX-heme-NO (see Fig. 1) display the same basic characteristics reported for six-coordinate ferrous nitrosylated heme-model compounds (e.g., Pip-FeTTP-NO) and heme-proteins (e.g., human adult Hb-NO obtained in the absence of allosteric effectors; i.e., R-state), showing an axial geometry (see Refs. [1,10,11,13–19]). Accordingly, electronic absorption spectroscopy data support that HPX-heme-NO is a six-coordinate heme-iron species [8]. Under conditions reported in the present study, heme binds to HDL, LDL, SA, and HPX mostly at the high affinity site(s), with no free heme present in solution (see Refs. [4,8,10–12]). No structural data of HDL-heme(-NO) and LDL-heme(NO) are currently available on which lipid components and protein clefts bind heme. Interestingly, several high and low affinity heme binding sites have been reported for HDL and LDL (see Refs. [4,20]). The ferric heme is located within the IB subdomain of SA which is constituted by the loop between the a-helices h8 and h9 and by the four contiguous a-helices h7, h8, h9, and h10. Residues Tyr138, Ile142, His145, Tyr161, and Lys190 show close interaction with the heme and appear to be contributors to the high heme affinity. Ferric heme is five coordinated with the Tyr161 phenolic oxygen atom [21]. Tyr161 becomes detached from the heme-iron atom upon NO binding to ferrous SA-heme, in the absence of allosteric effectors (i.e., N-form) (see Fig. 1 and Refs. [7,10,11]). Since no structural data of SA-heme-NO in the presence of drugs (i.e., B-form) are currently available, the protein fifth axial ligand of the heme iron atom cannot be identified. The heme high affinity site of HPX is located between Table 1 X-band EPR parameters of five-coordinate ferrous nitrosylated heme model compound and heme-proteins Species
A 3 (mT)
g1
g2
g3
FeTPP-NO a Human HDL-heme-NO b Human LDL-heme-NO b Human SA-heme-NO (N-form)c Human adult Hb-NO (T-state)d
1.73 1.68 1.69 1.65
2.102 2.098 2.100 2.095
2.064 2.058 2.060 2.060
2.010 2.009 2.010 2.010
1.63
2.094
2.051
2.009
a
From Ref. [15]. Present study. c From Refs. [10,11]. d From Ref. [14]. b
489
Table 2 X-band EPR parameters of six-coordinate ferrous nitrosylated heme model compound and heme-proteins Species a
Pip-FeTPP-NO Human SA-heme-NO (B-form)b Rabbit HPX-heme-NO c Human adult Hb-NO (R-state)d
A 3 (mT)
g1
g2
g3
2.17 u.s.
2.08 2.064
2.04 1.983
2.003 2.005
u.s. u.s.
2.068 2.060
1.988 1.986
2.005 2.006
a
From Ref. [15]. From Refs. [10,11]; u.s., unresolved signal. c Present study. d From Ref. [14]. b
the two orthogonal four-bladed b-propeller N- and Cdomains. In particular, ferric heme is six-coordinated with His213 and His266 residues. However, ferric heme could potentially be accommodated in a pocket at the broader end of the central tunnel of the N-domain of HPX, sixcoordinated to His82 and His127. Interestingly, the His213 residue of six-coordinate ferric HPX-heme became detached from the heme-iron atom upon cyanide binding (see Ref. [22]). Taken together, the results reported here show that HDL-heme-NO, LDL-heme-NO, and SA-heme-NO, in the absence of allosteric effectors (i.e., N-form), are fivecoordinate heme-iron complexes. By contrast, SA-hemeNO, in the presence of drugs (i.e., B-form), and HPXheme-NO are six-coordinate heme-iron systems (see Fig. 1, and Tables 1 and 2). Interestingly, the heme iron coordination state of HDL-heme-NO, LDL-heme-NO, SA-hemeNO, and HPX-heme-NO is in keeping with values of ferric heme dissociation rate constants which decrease in the following order: LDL.HDL.SA.HPX [4]. Therefore, it appears evident that interactions between the heme and the protein matrix are much stronger in HPX-heme-NO than in other heme-carriers, suggesting that HPX displays a more suitable heme binding cleft. Finally, the EPR-spectroscopic behavior of HDL-heme-NO, LDL-heme-NO, and HPXheme-NO may be helpful to investigate ligand binding and to highlight allosteric properties, as already reported for SA-heme-NO [10,11].
Abbreviations FeTTP-NO Hb Hb-NO HDL HDL-heme-NO HPX HPX-heme-NO LDL LDL-heme-NO
ferrous nitrosylated a,b,g,d-tetraphenylporphyrin hemoglobin ferrous nitrosylated Hb serum high density lipoproteins ferrous nitrosylated HDL-heme serum hemopexin ferrous nitrosylated HPX-heme serum low density lipoproteins ferrous nitrosylated LDL-heme
M. Fasano et al. / Journal of Inorganic Biochemistry 91 (2002) 487 – 490
490
NOR-1
Pip-FeTTP-NO SA SA-heme-NO SNAP
(6)-(E)-methyl-2-[(E)-hydroxyimino]6-methoxy-4-methyl-5-nitro-3-hexenamide piperidine-FeTTP-NO serum albumin ferrous nitrosylated SA-heme S-nitroso-N-acetyl-penicillamine
Acknowledgements Authors wish to thank Professor A. Desideri and Dr. M. Marino for helpful discussions and Mr. A. Merante for technical assistance. This work was partially supported by grants from the Ministry for Education, University, and Research of Italy (MIUR, Universita` ‘Roma Tre’—Fondi per lo Sviluppo 2000), as well as from the National Research Council of Italy (CNR, target-oriented project ‘Biotecnologie’).
References [1] H.F. Bunn, B.G. Forget, in: Hemoglobin: Molecular, Genetic and Clinical Aspects, W.B. Saunders, Philadelphia, PA, 1986. [2] T. Peters Jr., in: All about Albumin: Biochemistry, Genetics and Medical Applications, Academic Press, San Diego, CA, and London, 1996. [3] M.E. Conrad d, J.N. Umbreit, E.G. Moore, Am. J. Med. Sci. 318 (1999) 213–229.
[4] Y.I. Miller, N. Shaklai, Biochim. Biophys. Acta 1454 (1999) 153– 164. [5] M.E. Conrad, J.N. Umbreit, Am. J. Hematol. 64 (2000) 287–298. [6] J.R. Delanghe, M.R. Langlois, Clin. Chim. Acta 312 (2001) 13–23. [7] V.G. Kharitonov, V.S. Sharma, D. Magde, D. Koesling, Biochemistry 36 (1997) 6814–6818. [8] N. Shipulina, R.C. Hunt, N. Shaklai, A. Smith, J. Protein Chem. 17 (1998) 255–260. [9] P. Ascenzi, M. Colasanti, T. Persichini, M. Muolo, F. Polticelli, G. Venturini, D. Bordo, M. Bolognesi, Biol. Chem. 381 (2000) 623– 627. [10] S. Baroni, M. Mattu, A. Vannini, R. Cipollone, S. Aime, P. Ascenzi, M. Fasano, Eur. J. Biochem. 268 (2001) 6214–6220. [11] M. Mattu, A. Vannini, M. Coletta, M. Fasano, P. Ascenzi, J. Inorg. Biochem. 84 (2001) 293–296. [12] W.T. Morgan, P. Muster, F. Tatum, S.-m. Kao, J. Alam, A. Smith, J. Biol. Chem. 268 (1993) 6256–6262. [13] P. Ascenzi, M. Coletta, A. Desideri, M. Brunori, Biochim. Biophys. Acta 829 (1985) 299–302. [14] H. Kon, J. Biol. Chem. 243 (1968) 4350–4357. [15] B.B. Wayland, L.W. Olson, J. Am. Chem. Soc. 96 (1974) 6037– 6041. [16] A. Szabo, M.F. Perutz, Biochemistry 15 (1976) 4427–4428. [17] M.F. Perutz, Annu. Rev. Biochem. 48 (1979) 327–386. [18] W.E. Blumberg, Methods Enzymol. 76 (1981) 312–329. [19] M. Coletta, P. Ascenzi, M. Castagnola, B. Giardina, J. Mol. Biol. 249 (1995) 800–803. [20] G. Camejo, C. Halberg, A. Manschik-Lundin, E. Hurt-Camejo, B. Rosengren, H. Olsson, G.I. Hansson, G.B. Forsberg, B. Ylhen, J. Lipid. Res. 39 (1998) 755–766. [21] M. Wardell, Z. Wang, J.X. Ho, J. Robert, F. Ruker, J. Ruble, D.C. Carter, Biochem. Biophys. Res. Commun. 291 (2002) 813–819. [22] M. Paoli, B.F. Anderson, H.M. Baker, W.T. Morgan, A. Smith, E.N. Baker, Nat. Struct. Biol. 6 (1999) 926–931.
Short Communication
The heme-iron geometry of ferrous nitrosylated heme-serum lipoproteins, hemopexin, and albumin: a comparative EPR study a ,1
Mauro Fasano , Marco Mattu
b ,1
c b, , Massimo Coletta , Paolo Ascenzi *
a Department of Structural and Functional Biology, University of Insubria, Via Jean H. Dunant 3, I-21100 Varese, Italy Department of Biology and Interdepartmental Laboratory for Electron Microscopy, University ‘ Roma Tre’, Viale Guglielmo Marconi 446, I-00146 Rome, Italy c Department of Experimental Medicine and Biochemical Sciences, University of Rome ‘ Tor Vergata’, Via Montpellier 1, I-00133 Rome, Italy b
Received 5 March 2002; received in revised form 3 May 2002; accepted 24 May 2002 This study is dedicated to M.F. Perutz who pioneered studies on heme-proteins.
Abstract Serum high and low density lipoproteins, albumin, and hemopexin (HDL, LDL, SA, and HPX, respectively) serve as traps of toxic plasma heme and participate in its complete clearance by transportation to the liver. Moreover, SA-(heme) and HPX-heme have been proposed to facilitate NO scavenging in vivo. Here, the EPR-spectroscopic properties of ferrous nitrosylated heme-human high and low density lipoproteins (HDL-heme-NO and LDL-heme-NO, respectively) as well as of ferrous nitrosylated heme-rabbit serum hemopexin (HPX-heme-NO) are reported and analyzed in parallel with those of ferrous nitrosylated heme-human serum albumin (SA-heme-NO). HDL-heme-NO and LDL-heme-NO as well as SA-heme-NO, in the absence of allosteric effectors (i.e., N-form), are five-coordinate heme-iron species, characterized by the three-line splitting observed in the high magnetic field region of the X-band EPR spectrum. On the other hand, SA-heme-NO, in the presence of drugs (i.e., B-form), and HPX-heme-NO are six-coordinate heme-iron species, characterized by an X-band EPR spectrum with an axial geometry. The heme-iron coordination state of HDL-heme-NO, LDL-heme-NO, SA-heme-NO, and HPX-heme-NO is in keeping with values of ferric heme dissociation rate constants which decrease in the following order: LDL.HDL.SA.HPX. Altogether, these observations suggest that HPX displays a cleft much more suitable for heme binding than other heme-carriers. 2002 Elsevier Science Inc. All rights reserved. Keywords: Heme-human lipoproteins; Heme-rabbit hemopexin; Heme-human albumin; Ferrous nitrosylated heme-serum proteins; Heme-iron geometry; EPR spectroscopic properties
Plasma globin-free heme can stem from intracellular hemoglobin (Hb) degradation, which first leads to the accumulation of heme in red cell membrane followed by the heme dissociation from the membrane and binding to plasma proteins. Heme may also appear in plasma due to the heme release from extracellular Hb upon red cell lysis, under pathological conditions. Extracellular Hb tends to dissociate into dimers and the heme to oxidize to the ferric form which is then prone to be released from Hb to plasma (see Refs. [1–6]).
*Corresponding author. Tel.: 139-6-5517-6329; fax: 139-6-55176321. E-mail address: [email protected] (P. Ascenzi). 1 These authors contributed equally to this work.
Serum albumin, hemopexin as well as high and low density lipoproteins (SA, HPX, HDL, and LDL, respectively) serve as traps of the toxic plasma heme, thereby ensuring its complete clearance by transport to the liver (see Refs. [2–6]). Surprisingly, during the first seconds after heme appearance in plasma, more than 80% of this powerful oxidizer binds to HDL and LDL, the most oxidatively intolerant plasma components; and only the remaining 20% binds to antioxidative SA and HPX. SA and HPX slowly remove most of the heme from HDL and LDL (see Ref. [4]). Then, heme transits to HPX, that releases it into hepatic parenchymal cells only after internalization of HPX-heme by specific receptor-mediated endocytosis. After delivering the heme intracellularly, HPX is released intact into the bloodstream and the heme is degraded. Under conditions of severe hemolysis,
0162-0134 / 02 / $ – see front matter 2002 Elsevier Science Inc. All rights reserved. PII: S0162-0134( 02 )00473-7
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M. Fasano et al. / Journal of Inorganic Biochemistry 91 (2002) 487 – 490
hemopexin is overwhelmed and other processes come into play [3–6]. Binding of NO to SA-heme and HPX-heme suggested that these plasma heme-carriers may protect against NOmediated toxicity, especially in conditions of trauma and hemolysis. SA-heme and HPX-heme may facilitate NO scavenging by heme-iron chemistry. Moreover, SA-(heme) participates to NO metabolism by Cys34 (trans)-nitrosylation (see Refs. [2,7–9]). Here, the EPR-spectroscopic properties of ferrous nitrosylated heme-human high and low density lipoproteins as well as of ferrous nitrosylated heme-rabbit hemopexin (HDL-heme-NO, LDL-heme-NO, and HPX-heme-NO, respectively) are reported and analyzed in parallel with those of ferrous nitrosylated heme-human serum albumin (SA-heme-NO) (see Refs. [10,11]). Human serum HDL and LDL as well as heme chloride, NOR-1, and SNAP were obtained from Sigma (St. Louis, MO, USA). Rabbit serum HPX was prepared as previously reported (see Refs. [8,12]). Gaseous NO was purchased from Aldrich (Milwaukee, WI, USA). All the other products were purchased from Merck (Darmstadt, Germany). All chemicals were of analytical or reagent grade and used without further purification. HDL-heme (3.0310 25 M), LDL-heme (3.0310 25 M), and HPX-heme (2.8310 25 M) were prepared by adding 1.2-molar excess of HDL, LDL, and HPX to ferrous heme in 5.0310 22 M phosphate, pH 6.5 (HDL-heme and LDLheme) and 7.1 (HPX-heme); no free heme is present in solution (see Refs. [4,8,12]). The values of the dissociation equilibrium constant for heme binding to HDL, LDL, and HPX primary site(s) are lower than 10 29 M [4,8]. HDLheme-NO (3.0310 25 M), LDL-heme-NO (3.0310 25 M), and HPX-heme-NO (2.8310 25 M) were obtained, under anaerobic conditions, (i) by sequential addition of 10molar excess of sodium dithionite or sodium ascorbate and 4-molar excess of potassium nitrite to HDL-heme, LDLheme, and HPX-heme in 5.0310 22 M phosphate buffer, pH 6.5 or 7.1, (ii) by blowing purified NO over the HDL-heme, LDL-heme, and HPX-heme solution (5.03 10 22 M phosphate buffer, pH 6.5 or 7.1), in the absence and presence of 5-molar excess of sodium dithionite or sodium ascorbate, and (iii) by sequential addition of 10molar excess of dithiothreitol and 4-molar excess of NOR1 or SNAP to HDL-heme, LDL-heme, and HPX-heme in 5.0310 22 M phosphate buffer, pH 6.5 or 7.1 (see Refs. [7,8,10,11,13]). X-band EPR spectra of HDL-heme-NO, LDL-heme-NO, and HPX-heme-NO were collected at pH 6.5 or 7.1 (5.03 10 22 M phosphate buffer), and 110 K on a Bruker ESP 300 spectrometer with 100-kHz field modulation (see Refs. [10,11]). HDL-heme-NO, LDL-heme-NO, and HPX-hemeNO samples obtained by different methods gave identical X-band EPR spectra (data not shown). The X-band EPR spectra of HDL-heme-NO and LDLheme-NO as well as of SA-heme-NO, in the absence of
allosteric effectors (i.e., N-form) (see Fig. 1) display the same basic characteristics reported for five-coordinate ferrous nitrosylated heme-model compounds (e.g., a,b,g,dtetraphenylporphyrin (FeTTP-NO)) and heme-proteins (e.g., ferrous nitrosylated human adult hemoglobin (HbNO) obtained in the presence of allosteric effectors; i.e.,
Fig. 1. X-band EPR spectra of human HDL-heme-NO, human LDLheme-NO, human SA-heme-NO (in the absence of allosteric effectors and in the presence of drugs; i.e., N- and B-form, respectively), and rabbit HPX-heme-NO. X-band EPR spectra of SA-heme-NO were from Refs. [10,11]. X-band EPR spectra were obtained at pH 6.5 or 7.1, and 100 or 110 K. For further details, see text.
M. Fasano et al. / Journal of Inorganic Biochemistry 91 (2002) 487 – 490
T-state). Ferrous nitrosylated five-coordinate systems are characterized by the three-line splitting observed in the high magnetic field region of the X-band EPR spectrum (see Refs. [1,10,11,13–19]) (see Table 1). Interestingly, electronic absorption spectroscopy data support that SAheme-NO in the N-form is a five-coordinate heme-iron species [7]. The X-band EPR spectra of SA-heme-NO, in the presence of drugs (i.e., B-form), and HPX-heme-NO (see Fig. 1) display the same basic characteristics reported for six-coordinate ferrous nitrosylated heme-model compounds (e.g., Pip-FeTTP-NO) and heme-proteins (e.g., human adult Hb-NO obtained in the absence of allosteric effectors; i.e., R-state), showing an axial geometry (see Refs. [1,10,11,13–19]). Accordingly, electronic absorption spectroscopy data support that HPX-heme-NO is a six-coordinate heme-iron species [8]. Under conditions reported in the present study, heme binds to HDL, LDL, SA, and HPX mostly at the high affinity site(s), with no free heme present in solution (see Refs. [4,8,10–12]). No structural data of HDL-heme(-NO) and LDL-heme(NO) are currently available on which lipid components and protein clefts bind heme. Interestingly, several high and low affinity heme binding sites have been reported for HDL and LDL (see Refs. [4,20]). The ferric heme is located within the IB subdomain of SA which is constituted by the loop between the a-helices h8 and h9 and by the four contiguous a-helices h7, h8, h9, and h10. Residues Tyr138, Ile142, His145, Tyr161, and Lys190 show close interaction with the heme and appear to be contributors to the high heme affinity. Ferric heme is five coordinated with the Tyr161 phenolic oxygen atom [21]. Tyr161 becomes detached from the heme-iron atom upon NO binding to ferrous SA-heme, in the absence of allosteric effectors (i.e., N-form) (see Fig. 1 and Refs. [7,10,11]). Since no structural data of SA-heme-NO in the presence of drugs (i.e., B-form) are currently available, the protein fifth axial ligand of the heme iron atom cannot be identified. The heme high affinity site of HPX is located between Table 1 X-band EPR parameters of five-coordinate ferrous nitrosylated heme model compound and heme-proteins Species
A 3 (mT)
g1
g2
g3
FeTPP-NO a Human HDL-heme-NO b Human LDL-heme-NO b Human SA-heme-NO (N-form)c Human adult Hb-NO (T-state)d
1.73 1.68 1.69 1.65
2.102 2.098 2.100 2.095
2.064 2.058 2.060 2.060
2.010 2.009 2.010 2.010
1.63
2.094
2.051
2.009
a
From Ref. [15]. Present study. c From Refs. [10,11]. d From Ref. [14]. b
489
Table 2 X-band EPR parameters of six-coordinate ferrous nitrosylated heme model compound and heme-proteins Species a
Pip-FeTPP-NO Human SA-heme-NO (B-form)b Rabbit HPX-heme-NO c Human adult Hb-NO (R-state)d
A 3 (mT)
g1
g2
g3
2.17 u.s.
2.08 2.064
2.04 1.983
2.003 2.005
u.s. u.s.
2.068 2.060
1.988 1.986
2.005 2.006
a
From Ref. [15]. From Refs. [10,11]; u.s., unresolved signal. c Present study. d From Ref. [14]. b
the two orthogonal four-bladed b-propeller N- and Cdomains. In particular, ferric heme is six-coordinated with His213 and His266 residues. However, ferric heme could potentially be accommodated in a pocket at the broader end of the central tunnel of the N-domain of HPX, sixcoordinated to His82 and His127. Interestingly, the His213 residue of six-coordinate ferric HPX-heme became detached from the heme-iron atom upon cyanide binding (see Ref. [22]). Taken together, the results reported here show that HDL-heme-NO, LDL-heme-NO, and SA-heme-NO, in the absence of allosteric effectors (i.e., N-form), are fivecoordinate heme-iron complexes. By contrast, SA-hemeNO, in the presence of drugs (i.e., B-form), and HPXheme-NO are six-coordinate heme-iron systems (see Fig. 1, and Tables 1 and 2). Interestingly, the heme iron coordination state of HDL-heme-NO, LDL-heme-NO, SA-hemeNO, and HPX-heme-NO is in keeping with values of ferric heme dissociation rate constants which decrease in the following order: LDL.HDL.SA.HPX [4]. Therefore, it appears evident that interactions between the heme and the protein matrix are much stronger in HPX-heme-NO than in other heme-carriers, suggesting that HPX displays a more suitable heme binding cleft. Finally, the EPR-spectroscopic behavior of HDL-heme-NO, LDL-heme-NO, and HPXheme-NO may be helpful to investigate ligand binding and to highlight allosteric properties, as already reported for SA-heme-NO [10,11].
Abbreviations FeTTP-NO Hb Hb-NO HDL HDL-heme-NO HPX HPX-heme-NO LDL LDL-heme-NO
ferrous nitrosylated a,b,g,d-tetraphenylporphyrin hemoglobin ferrous nitrosylated Hb serum high density lipoproteins ferrous nitrosylated HDL-heme serum hemopexin ferrous nitrosylated HPX-heme serum low density lipoproteins ferrous nitrosylated LDL-heme
M. Fasano et al. / Journal of Inorganic Biochemistry 91 (2002) 487 – 490
490
NOR-1
Pip-FeTTP-NO SA SA-heme-NO SNAP
(6)-(E)-methyl-2-[(E)-hydroxyimino]6-methoxy-4-methyl-5-nitro-3-hexenamide piperidine-FeTTP-NO serum albumin ferrous nitrosylated SA-heme S-nitroso-N-acetyl-penicillamine
Acknowledgements Authors wish to thank Professor A. Desideri and Dr. M. Marino for helpful discussions and Mr. A. Merante for technical assistance. This work was partially supported by grants from the Ministry for Education, University, and Research of Italy (MIUR, Universita` ‘Roma Tre’—Fondi per lo Sviluppo 2000), as well as from the National Research Council of Italy (CNR, target-oriented project ‘Biotecnologie’).
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