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Copyright © 2014 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.
Thoughts and Progress Usefulness of Myoglobin Containing Cobalt Heme Cofactor in Designing a Myoglobin-Based Artificial Oxygen Carrier *Saburo Neya, †Takashi Yonetani, and ‡Akira T. Kawaguchi *Department of Physical Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan; †Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA; and ‡School of Medicine, Tokai University, Isehara, Kanagawa, Japan Abstract: The structure and reactivity of cobalt-replaced myoglobin (Mb) were investigated to explore its possible application as an artificial oxygen carrier. Ligand binding analysis with relaxation kinetics revealed that various ligands bind to Co(III) Mb, contrary to the earlier thoughts. The equilibration process, however, was so slow that it proceeded over 90 min. These characteristic profiles of oxidized Co(III) Mb were ascribed to the electronic structure of Co(III) ion which is one electron larger than Fe(III) ion. The oxygen affinity of reduced Co(II) Mb was much smaller than that of Fe(II) Mb indicating that Co(II) Mb has excellent oxygen transport ability. The latter observation, together with the lack of carbon monoxide binding in Co(II) Mb, suggests utility of Co(II) Mb as Mb-based oxygen carriers. The present results on cobalt-substituted Mb are useful in designing myoglobin-based oxygen carriers. Key Words: Myoglobin—Metal replacement— Cobalt heme—Ligand binding—Artificial oxygen carrier.
An artificial oxygen carrier is an important component in blood transfusion. A number of candidates such as perfluorocarbon emulsions, hemoglobin (Hb)-based oxygen carriers, liposome-encapsulated and recombinant Hbs have been investigated (1). Myoglobin (Mb) is another class of oxygen-binding
doi:10.1111/aor.12327 Received December 2013; revised March 2014. Address correspondence and reprint requests to Prof. Saburo Neya, Department of Physical Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, Inage-Yayoi, Chiba 263-8522, Japan. E-mail: [email protected] Presented in part at the 4th International Symposium on Artificial Oxygen Carriers, held September 28, 2013 in Yokohama, Japan.
hemoprotein. However, its application to the oxygen carrier has been almost undeveloped. This is because the oxygen affinity of Mb is so high that it does not effectively release bound oxygen under physiological conditions. We have been exploring the Mb-based oxygen carrier by controlling the high oxygen affinity of Mb (2,3). Alteration of the molecular shape and planarity of heme successfully reduced the oxygen affinity and increased the oxygen-carrying capacity of Mb. For instance, the P50 = 0.07 mm Hg, the partial oxygen pressure at half saturation of Fe(II) Mb, was increased by 25-fold to 1.8 mm Hg on the nonplanar heme deformation (4). When ferrous corrphycene, a trapezoidal heme isomer, was coupled with Mb, a P50 = 37 mm Hg was observed (5). The value is comparable with P50 = 32 mm Hg of red blood cells (6). These results indicate that heme chemical modification is effective to control the oxygen affinity of Mb. Heme iron (Fe) is the key component for the normal function of Hb and Mb. Replacement of the Fe ion with other metal ions such as Mn, Ni, Cu, and Co has been extensively examined to perturb the Fe-histidine bond and to modify the physiological properties of hemoproteins (7). Among the metal ions investigated, Co is of particular interest because Co(II)-containing organic compounds such as Schiff bases and porphyrins are capable of reversible oxygenation (8). The Co(II)-substituted hemoproteins have been the subject of extensive spectroscopic analysis because the oxygenated compounds are paramagnetic to allow detailed examination with, for example, electron spin resonance (EPR) (9,10). This is in contrast with ferrous oxy derivatives of Hb and Mb, which are diamagnetic and EPR invisible. In contrast, the reaction of oxidized Co(III) Mb with ligand has been only partly investigated. In the present article, we will first characterize the reactivity of Co(III) Mb, and then discuss the utility of Co(II) Mb as a Mb-based artificial oxygen carrier. The results on Co(II) Mb will be helpful to develop new artificial oxygen carriers which have been mainly developed with Hb-related materials. MATERIALS AND METHODS Cobalt-containing porphyrins were synthesized according to the literature (10,11). The Co(III) hemes were coupled with apoMb, prepared from Artificial Organs 2014, 38(8):715–719
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sperm whale Mb (Sigma Chemicals, St. Louis, MO, USA), and purified with chromatography on cellulose column (Whatman, CM52) (11). The ligand binding to the reconstituted Co(III) Mb was monitored on a Shimadzu MPS-2000 spectrophotometer (Kyoto, Japan) equipped with a thermostatted sample holder at 20°C. The difference spectra were obtained by subtraction of the initial spectrum, saved in a computer memory of the spectrophotometer, from the observed curve. The ligand binding to Co(III) Mb was so slow that the relaxation process could be monitored with a conventional spectrophotometer, Shimadzu MPS2000, after addition of 1–20-mM of ligand to about 10 μM of the Co(III) Mb solution. The relaxation curves of ligand binding were analyzed on the basis of a simple 1:1 complex formation according to the method by Bernasconi (12). Reduced Mb Co(II) was obtained by the coupling of apoMb and dithionite-reduced Co(II) heme under an anaerobic condition. Excess of pyridine and sodium dithionite was removed after passing through a Sephadex G-29 column, as we reported previously (10). The oxygen affinity was measured with Imai’s automatic recording apparatus in the presence of an enzymic reducing system (10). The ligand binding for Co-substituted Mbs was measured more than twice by spectrophotometry. The experimental errors in the binding constants were estimated with the numerical data reduction procedures (13). RESULTS Reactivity of Co(III) Mb The reactivity of oxidized Co(III) Mb has been scarcely characterized. Rinsdale and coworkers reported in 1973 a characteristic visible spectrum with dominant α and β absorption bands for Co(III) Hb (14). The spectrum was attributed to an internal hemichrome structure coordinated with both the proximal and distal histidines to the Co(III) center. They also reported that only strongly coordinating CN- replaces the distal histidine to form the CNcomplex. It seems that a similar view has been long applied to Co(III) Mb because Mb and Hb in general adopt the same coordination structure. However, the X-ray crystallographic analysis by Brucker et al. revealed in 1996 that the sixth ligand in Co(III) Mb is not the distal histidine but a water molecule (15). The result indicates that the apparent inertness of Co(III) Mb is not ascribed to the hemichrome structure. We accordingly analyzed in detail the ligand binding of Co(III) Mb. Artif Organs, Vol. 38, No. 8, 2014
The visible spectrum of Co(III) Mb in Fig. 1A exhibited a Soret band with ε414 = 124 1/(mM cm) as well as the sharp α and β bands at 552 (ε = 9.5 1/ (mM cm)) and 525 (ε = 11.4 1/(mM cm)) nm, respectively. Increase in pH caused a spectral transition with well-defined isosbestic points to reflect the dissociation of Co(III)-bound H2O to OH- at pH = 8.56 (11). The latter observation suggests that the Co(III)bound H2O is hydrogen-bonded with the distal histidine (15). It is to be pointed out that the α and β bands of aquomet Co(III) Mb (Fig. 1A) are rather intense. This is in contrast with aquomet Fe(III) Mb spectrum where the α (ε580 = 2.5 1/(mM cm)) and β (ε530 = 7.5 1/(mM cm)) peaks are observed as obscure inflectional bands (16). Because a good correlation between the appearance of the α and β bands and the dominance of low-spin state of the metal center is well established in hemoprotein (16), the visible spectrum in Fig. 1A is indicative of the low-spin (S = 0) state for Co(III) Mb. We measured the proton nuclear magnetic resonance (NMR) spectrum of Co(III) Mb containing deuteroheme to identify the spin state of Co(III) Mb. The NMR spectrum showed no paramagnetic signals from the heme 2,4protons which should appear in the 50–60 ppm region if it were high-spin (result not shown). The observation confirmed the low-spin of aquomet Co(III) Mb, in contrast with aquomet Fe(III) Mb which is high-spin (S = 5/2) (16). We examined subsequently the ligand behavior of Co(III) Mb, and found that imidazole, a weakly coordinating ligand to hemoproteins, binds to the Mb. The Soret absorption transition induced by imidazole is illustrated in Fig. 1B. The spectrum changed over 70 min with clear isosbestic points at 360, 414, and 436 nm in the presence of imidazole (Fig. 1C,D). Analysis of the 418-nm absorption in Fig. 1D with the method (12,13) afforded a relaxation rate 1/τ = (4.55 ± 0.09) × 10−2/min at 18 mM of imidazole. With other 1/τvalues obtained at lower imidazole concentrations, we obtained the kinetic and equilibrium constants as shown in Table 1. It is notable in Table 1 that the rate constants for Co(III) Mb are significantly smaller than those in Fe(III) Mb (17) while the equilibrium constants are comparable with each other. For pyridine, N3-, SCN-, and CN-, the ligand affinities K of (9.6 ± 2.9) × 102, (3.7 ± 1.5) × 102, (9.3 ± 3.0) × 103, (2.8 ± 1.0) × 104 M−1, respectively, were also obtained. These K values were comparable with those found in Fe(III) Mb. However, a tendency in the kinetic parameters, that is, much smaller kon and koff in comparison with those in Fe(III) Mb, was very characteristic of Co(III) Mb.
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FIG. 1. Electronic absorption spectra of Co(III) Mb containing deuteroheme. (A) Visible absorption spectrum of aquomet Mb in 0.1-M Tris buffer at pH 7.0 and 20°C. (B) ), the Soret bands of aquomet Mb ( imidazole complex ( ), and the difference spectrum ( ) in 0.1 M Tris buffer at pH 7.0 and 20°C. The spectrum of the imidazole complex was recorded 120 min after adding 3.0 mM of imidazole. (C) Soret absorption transition of Co(III) Mb after addition of 17.8 mM imidazole in 0.1 M Tris buffer at pH 7.0 and 20°C. (D) Time course of the 418 nm peak in (C).
Oxygen binding to Co(II) Mb The coordination structures of oxy and deoxy Co(II) Mb are identical to those of the corresponding Fe(II) Mb derivatives (15). We have measured the oxygen affinities of the Co(II) Mbs bearing several hemes (10). The P50 values for oxygen of proto-,
meso-, and deuteroheme Co(II) Mbs, taken from our previous report (10), were 50.0, 50.0, and 20.0 mm Hg, respectively, at 23°C (Fig. 2). However, the P50 values of Fe(II) Mbs containing proto(1.0 mm Hg), meso- (0.54 mm Hg), or deuteroheme (0.21 mm Hg) at the same condition were much
TABLE 1. Equilibrium and kinetic parameters of imidazole binding to Co(III) and Fe(III) Mb Materials Co(III) Mb Fe(III) Mb
kon (M/s)
koff (per second)
(4.4 ± 0.6) × 10 2.8 × 102
–2
(9.1 ± 0.8) × 10 4.5
–6
K (per M)
Reference
(4.8 × 1.1) × 10 62
2
This work (17)
Condition, pH 7.0 and 20°C for Co(III) Mb and pH 7.1 and 21–23°C for Fe(III) Mb.
FIG. 2. Structure of the prosthetic groups in Co(II) Mb. The values of P50 for O2 at 23°C are from our previous results (10).
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smaller (10). Thus, Co(II) Mbs exhibit lower oxygen affinity than Fe(II) Mbs. Generally, the P50 values in Co(II) Mbs were nearly comparable with or larger than the P50 = 10 mm Hg of Fe(II) Hb (10) and P50 = 32 mm Hg of red blood cells (6). The small P50 in deuteroheme Co(II) Mb among the three Mbs in Fig. 2 was ascribed to the decreased heme–globin interactions due to smaller bulk of the hydrogen atoms at the 2,4-positions of porphyrin (10). DISCUSSION Characteristic reactivity of Co(III) Mb The low reactivity of Co(III) Hb with CN- has been ascribed to an internal hemichrome structure. The same structural analogy seems to have been applied to Co(III) Mb. Our analysis, however, supported an aquomet structure for Co(III) Mb (15), and further suggested that the magnitude of hydrogen bonding between H2O and the distal histidine in Co(III) Mb is almost identical to that in aquomet Fe(III) Mb (11). We further found that various weak ligands coordinate to Co(III) Mb with rather high affinities. It is notable in Table 1 that the imidazole affinity to Co(III) Mb is moderately high, and that the association constant kon of imidazole is much smaller than that of Fe(III) Mb (17). The same holds for the kon and koff of the ligand in N3−, CN−, SCN−, and pyridine (11). The small kon constant observed for imidazole (Table 1) may be explained by the distinct electronic charge of Co(III) ion with six 3d-electrons, which is one electron larger than the Fe(III) ion with five 3d-electrons. The larger negative charge on the Co(III) ion could retard association of the unshared electron pair on imidazole. In addition, the Co(III)-N(imidazole) bond is shorter than the Fe(III)-N(imidazole) bond. For instance, Co(III)´˚ ) (18) is slightly comN(imidazole) bond (1.927 A pressed in comparison with the Fe(III)-N(imidazole) ´˚ ) (19) in the bis-imidazole adducts of bond (1.977 A metallo porphyrins. Interestingly, Nakamura demonstrated from the NMR analysis that the slightly shorter Co(III)-N(imidazole) bond in the Co(III) heme dramatically (approx. 1/105 fold) lowered the dissociation rate of the coordinating imidazole (20). Thus, the small koff rate in Co(III) Mb in Table 1 is reasonably explained in terms of the stronger Co(III)-imidazole bond than the corresponding bond in Fe(III) Mb. Utility of reduced Co(II) Mb as an Mb-based oxygen carrier The X-ray analysis of deoxy Co(II) Mb revealed that the Co(II) ion is five-coordinate (15). One Artif Organs, Vol. 38, No. 8, 2014
notable feature of Co(II) Mb, relative to Fe(II) Mb, is the much lower oxygen affinity. The P50 values of Co(II) Mbs bearing proto-, meso-, and deuterohemes are 50 to 100-fold larger than those of Fe(II) Mbs (Fig. 2). Much lowered oxygen affinities of Co(II) Mbs indicate that Fe(II) Mb is converted from the reservoir to carrier for oxygen after the metal substitution. With the physiological partial oxygen pressures in arterial (100 mm Hg) and venous (40 mm Hg) blood, we calculated the high transporting ability of oxygen in Co(II) Mbs with proto-, meso-, and deuteroheme to be 23, 23, and 50%, respectively (Fig. 2). It is well known that nitric oxide (NO) is a physiologic messenger molecule, and it has a high affinity to Hb. Possible NO capture by Co(II) Mb may induce vasoconstriction and increase blood pressure in the treated animals. The NO affinity of Co(II) Mb is the matter of significance. Rapson et al. reported the NO dissociation constants of 32 μM and 10 μM for Fe(II) Mb and Co(II) Mb, respectively (21), implying that the two Mbs have comparable NO affinities. A resonance Raman spectroscopic study indicated that the Co(II)-NO linkage is weaker than the Fe(II)-NO bond, owing to the steric constraint in the Co(II)-NO (22). The weaker Co(II)-NO bond may suggest a reduced NO affinity in Co(II) Mb; however, Co(II) Mb and Fe(II) Mb exhibit similar NO affinities (21). The similar NO affinities, in turn, indicate that Co(II) Mb has a larger kon rate of NO binding than Fe(II) Mb. This is consistent with the observation in ligand kinetics that Co(II) Mb (30 μM/s) has a larger kon rate than Fe(II) Mb (20 μM/s) (23). It is now widely accepted that carbon monoxide (CO) acts as a second messenger next to NO. Physiologic doses of CO, produced upon heme degradation by heme oxygenase, protect cells through potential anti-inflammatory, antiproliferative, or anti-apoptotic effects. Wang and coworkers recently demonstrated that CO is therapeutic in ischemiareperfusion brain injury (24). CO does not bind to Co(II) Mb at all while it has a high affinity to Fe(II) Mb. The lack of CO binding ability in Co(II) Mb is notable as it does not remove the physiological CO. CONCLUSION In summary, the present work provides an overview on the reactivity of cobalt-bearing myoglobin in the Co(III) and Co(II) states. Co(III) Mb exhibits characteristic spectroscopic and ligand-binding profiles despite that it has a common coordination structure with aquomet Fe(III) Mb. Co(II) Mb, however,
THOUGHTS AND PROGRESS has properties to show no CO affinity and low oxygen binding. The latter observation demonstrates a potential utility of Co(II)-substituted Mb as an oxygen carrier. Accordingly, Co(II) Mb is an attractive material to develop the Mb-based artificial oxygen carriers. Acknowledgments: This work was supported by Grants-in-Aid for the Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan, Nos. 23590040 (C) (to S.N.) and 24249086 (A) (to A.T.K.). We also thank the Yonetani Research Fund No. 2013 for the financial support to T.Y. REFERENCES 1. Kim HW, Greenburg AG. Artificial oxygen carriers as red blood cell substitutes: a selected review and current status. Artif Organs 2004;58:813–28. 2. Neya S, Suzuki M, Hoshino T. Novel controlling mechanism of oxygen affinity in myoglobin with isomeric porphyrins. Artif Organs 2009;33:189–93. 3. Neya S, Kawaguchi AT. Inherently distorted heme as a novel tool for myoglobin-based oxygen carrier. Artif Organs 2012; 36:220–3. 4. Neya S, Suzuki M, Hoshino T, et al. Molecular insight into intrinsic heme distortion in ligand binding in hemoprotein. Biochemistry 2010;49:5642–50. 5. Neya S, Funasaki N, Hori H, et al. Functional regulation of myoglobin by iron corrphycene. Chem Lett 1999;28:989–90. 6. Villela NR, Cabrales P, Tsai AG, Intaglietta M. Microcirculatory effects of changing blood hemoglobin oxygen affinity during hemorrhagic shock resuscitation in an experimental model. Shock 2009;31:645–52. 7. Dickinson LC. Metal replaced hemoproteins. J Chem Educ 1976;53:381–5. 8. Basolo F, Hoffman BM, Ibers JA. Synthetic oxygen carriers of biological interest. Acc Chem Res 1975;8:384–92.
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9. Chien JCW, Dickinson LC. Electron paramagnetic resonance of single crystal oxycobalt myoglobin and deoxy myoglobin. Proc Natl Acad Sci USA 1972;69:2783–7. 10. Yonetani T, Yamamoto H, Woodraw GV III. Studies on cobalt myoglobins and hemoglobins. J Biol Chem 1974;249: 682–90. 11. Neya S, Suzuki M, Hoshino T, Kawaguchi AT. Relaxation analysis of ligand binding to the myoglobin reconstituted with cobaltic heme. Inorg Chem 2013;52:7387–93. 12. Bernasconi CF. Relaxation Kinetics. New York: Academic Press, 1976;11–9. 13. Mortimer RG. Mathematics for Physical Chemistry. New York: Macmillan, 1981;266–309. 14. Rinsdale S, Cassatt JC, Steinhardt J. Acid denaturation studies on a cobalt(III) protoporphyrin-globin complex. J Biol Chem 1973;248:771–6. 15. Brucker EA, Olson JS, Phillips GN Jr, Dou Y, Ikeda-Saito M. High resolution crystal structure of the deoxy, oxy, and aquomet forms of cobalt myoglobin. J Biol Chem 1996;271:25419–22. 16. Brill AS, Williams RJP. The absorption spectra, magnetic moments and the binding of iron in some hemoproteins. Biochem J 1961;78:246–53. 17. Antonini E, Brunori M. Hemoglobin and Myoglobin in Their Reactions with Ligands. London: North-Holland Publishing, 1971;230. 18. Lauher JW, Ibers JA. Stereochemistry of cobalt porphyrins II. J Am Chem Soc 1974;96:4447–52. 19. Collins DM, Countryman R, Hoard JL. Stereochemistry of low spin iron porphyrins I. J Am Chem Soc 1972;94:2066–72. 20. Nakamura M. Effects of ortho-methyl substituents on the dissociation rate of imidazole ligand in tetraarylporphyrinatocobalt(III) complexes. Bull Chem Soc Jpn 1995;68:197–203. 21. Rapson TD, Dacres H, Trowell SC. Fluorescent nitric oxide detection using cobalt substituted myoglobin. Roy Soc Chem Adv 2014;4:10269–72. 22. Hu S. Resonance Raman spectroscopic studies of the nitric oxide adducts of cobaltous-reconstituted myoglobin and hemoglobin. Inorg Chem 1993;32:1081–5. 23. Quillin ML, Li T, Olson JS, et al. Structural and functional effects of apolar mutations of the distal valine in myoglobin. J Mol Biol 1995;245:416–36. 24. Wang B, Cao W, Biswal S, Doré S. Carbon monoxideactivated Nrf2 pathway leads to protecting against permanent focal cerebral ischemia. Stroke 2011;42:2605–10.
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Copyright © 2014 International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.
Thoughts and Progress Usefulness of Myoglobin Containing Cobalt Heme Cofactor in Designing a Myoglobin-Based Artificial Oxygen Carrier *Saburo Neya, †Takashi Yonetani, and ‡Akira T. Kawaguchi *Department of Physical Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan; †Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, PA, USA; and ‡School of Medicine, Tokai University, Isehara, Kanagawa, Japan Abstract: The structure and reactivity of cobalt-replaced myoglobin (Mb) were investigated to explore its possible application as an artificial oxygen carrier. Ligand binding analysis with relaxation kinetics revealed that various ligands bind to Co(III) Mb, contrary to the earlier thoughts. The equilibration process, however, was so slow that it proceeded over 90 min. These characteristic profiles of oxidized Co(III) Mb were ascribed to the electronic structure of Co(III) ion which is one electron larger than Fe(III) ion. The oxygen affinity of reduced Co(II) Mb was much smaller than that of Fe(II) Mb indicating that Co(II) Mb has excellent oxygen transport ability. The latter observation, together with the lack of carbon monoxide binding in Co(II) Mb, suggests utility of Co(II) Mb as Mb-based oxygen carriers. The present results on cobalt-substituted Mb are useful in designing myoglobin-based oxygen carriers. Key Words: Myoglobin—Metal replacement— Cobalt heme—Ligand binding—Artificial oxygen carrier.
An artificial oxygen carrier is an important component in blood transfusion. A number of candidates such as perfluorocarbon emulsions, hemoglobin (Hb)-based oxygen carriers, liposome-encapsulated and recombinant Hbs have been investigated (1). Myoglobin (Mb) is another class of oxygen-binding
doi:10.1111/aor.12327 Received December 2013; revised March 2014. Address correspondence and reprint requests to Prof. Saburo Neya, Department of Physical Chemistry, Graduate School of Pharmaceutical Sciences, Chiba University, Inage-Yayoi, Chiba 263-8522, Japan. E-mail: [email protected] Presented in part at the 4th International Symposium on Artificial Oxygen Carriers, held September 28, 2013 in Yokohama, Japan.
hemoprotein. However, its application to the oxygen carrier has been almost undeveloped. This is because the oxygen affinity of Mb is so high that it does not effectively release bound oxygen under physiological conditions. We have been exploring the Mb-based oxygen carrier by controlling the high oxygen affinity of Mb (2,3). Alteration of the molecular shape and planarity of heme successfully reduced the oxygen affinity and increased the oxygen-carrying capacity of Mb. For instance, the P50 = 0.07 mm Hg, the partial oxygen pressure at half saturation of Fe(II) Mb, was increased by 25-fold to 1.8 mm Hg on the nonplanar heme deformation (4). When ferrous corrphycene, a trapezoidal heme isomer, was coupled with Mb, a P50 = 37 mm Hg was observed (5). The value is comparable with P50 = 32 mm Hg of red blood cells (6). These results indicate that heme chemical modification is effective to control the oxygen affinity of Mb. Heme iron (Fe) is the key component for the normal function of Hb and Mb. Replacement of the Fe ion with other metal ions such as Mn, Ni, Cu, and Co has been extensively examined to perturb the Fe-histidine bond and to modify the physiological properties of hemoproteins (7). Among the metal ions investigated, Co is of particular interest because Co(II)-containing organic compounds such as Schiff bases and porphyrins are capable of reversible oxygenation (8). The Co(II)-substituted hemoproteins have been the subject of extensive spectroscopic analysis because the oxygenated compounds are paramagnetic to allow detailed examination with, for example, electron spin resonance (EPR) (9,10). This is in contrast with ferrous oxy derivatives of Hb and Mb, which are diamagnetic and EPR invisible. In contrast, the reaction of oxidized Co(III) Mb with ligand has been only partly investigated. In the present article, we will first characterize the reactivity of Co(III) Mb, and then discuss the utility of Co(II) Mb as a Mb-based artificial oxygen carrier. The results on Co(II) Mb will be helpful to develop new artificial oxygen carriers which have been mainly developed with Hb-related materials. MATERIALS AND METHODS Cobalt-containing porphyrins were synthesized according to the literature (10,11). The Co(III) hemes were coupled with apoMb, prepared from Artificial Organs 2014, 38(8):715–719
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sperm whale Mb (Sigma Chemicals, St. Louis, MO, USA), and purified with chromatography on cellulose column (Whatman, CM52) (11). The ligand binding to the reconstituted Co(III) Mb was monitored on a Shimadzu MPS-2000 spectrophotometer (Kyoto, Japan) equipped with a thermostatted sample holder at 20°C. The difference spectra were obtained by subtraction of the initial spectrum, saved in a computer memory of the spectrophotometer, from the observed curve. The ligand binding to Co(III) Mb was so slow that the relaxation process could be monitored with a conventional spectrophotometer, Shimadzu MPS2000, after addition of 1–20-mM of ligand to about 10 μM of the Co(III) Mb solution. The relaxation curves of ligand binding were analyzed on the basis of a simple 1:1 complex formation according to the method by Bernasconi (12). Reduced Mb Co(II) was obtained by the coupling of apoMb and dithionite-reduced Co(II) heme under an anaerobic condition. Excess of pyridine and sodium dithionite was removed after passing through a Sephadex G-29 column, as we reported previously (10). The oxygen affinity was measured with Imai’s automatic recording apparatus in the presence of an enzymic reducing system (10). The ligand binding for Co-substituted Mbs was measured more than twice by spectrophotometry. The experimental errors in the binding constants were estimated with the numerical data reduction procedures (13). RESULTS Reactivity of Co(III) Mb The reactivity of oxidized Co(III) Mb has been scarcely characterized. Rinsdale and coworkers reported in 1973 a characteristic visible spectrum with dominant α and β absorption bands for Co(III) Hb (14). The spectrum was attributed to an internal hemichrome structure coordinated with both the proximal and distal histidines to the Co(III) center. They also reported that only strongly coordinating CN- replaces the distal histidine to form the CNcomplex. It seems that a similar view has been long applied to Co(III) Mb because Mb and Hb in general adopt the same coordination structure. However, the X-ray crystallographic analysis by Brucker et al. revealed in 1996 that the sixth ligand in Co(III) Mb is not the distal histidine but a water molecule (15). The result indicates that the apparent inertness of Co(III) Mb is not ascribed to the hemichrome structure. We accordingly analyzed in detail the ligand binding of Co(III) Mb. Artif Organs, Vol. 38, No. 8, 2014
The visible spectrum of Co(III) Mb in Fig. 1A exhibited a Soret band with ε414 = 124 1/(mM cm) as well as the sharp α and β bands at 552 (ε = 9.5 1/ (mM cm)) and 525 (ε = 11.4 1/(mM cm)) nm, respectively. Increase in pH caused a spectral transition with well-defined isosbestic points to reflect the dissociation of Co(III)-bound H2O to OH- at pH = 8.56 (11). The latter observation suggests that the Co(III)bound H2O is hydrogen-bonded with the distal histidine (15). It is to be pointed out that the α and β bands of aquomet Co(III) Mb (Fig. 1A) are rather intense. This is in contrast with aquomet Fe(III) Mb spectrum where the α (ε580 = 2.5 1/(mM cm)) and β (ε530 = 7.5 1/(mM cm)) peaks are observed as obscure inflectional bands (16). Because a good correlation between the appearance of the α and β bands and the dominance of low-spin state of the metal center is well established in hemoprotein (16), the visible spectrum in Fig. 1A is indicative of the low-spin (S = 0) state for Co(III) Mb. We measured the proton nuclear magnetic resonance (NMR) spectrum of Co(III) Mb containing deuteroheme to identify the spin state of Co(III) Mb. The NMR spectrum showed no paramagnetic signals from the heme 2,4protons which should appear in the 50–60 ppm region if it were high-spin (result not shown). The observation confirmed the low-spin of aquomet Co(III) Mb, in contrast with aquomet Fe(III) Mb which is high-spin (S = 5/2) (16). We examined subsequently the ligand behavior of Co(III) Mb, and found that imidazole, a weakly coordinating ligand to hemoproteins, binds to the Mb. The Soret absorption transition induced by imidazole is illustrated in Fig. 1B. The spectrum changed over 70 min with clear isosbestic points at 360, 414, and 436 nm in the presence of imidazole (Fig. 1C,D). Analysis of the 418-nm absorption in Fig. 1D with the method (12,13) afforded a relaxation rate 1/τ = (4.55 ± 0.09) × 10−2/min at 18 mM of imidazole. With other 1/τvalues obtained at lower imidazole concentrations, we obtained the kinetic and equilibrium constants as shown in Table 1. It is notable in Table 1 that the rate constants for Co(III) Mb are significantly smaller than those in Fe(III) Mb (17) while the equilibrium constants are comparable with each other. For pyridine, N3-, SCN-, and CN-, the ligand affinities K of (9.6 ± 2.9) × 102, (3.7 ± 1.5) × 102, (9.3 ± 3.0) × 103, (2.8 ± 1.0) × 104 M−1, respectively, were also obtained. These K values were comparable with those found in Fe(III) Mb. However, a tendency in the kinetic parameters, that is, much smaller kon and koff in comparison with those in Fe(III) Mb, was very characteristic of Co(III) Mb.
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FIG. 1. Electronic absorption spectra of Co(III) Mb containing deuteroheme. (A) Visible absorption spectrum of aquomet Mb in 0.1-M Tris buffer at pH 7.0 and 20°C. (B) ), the Soret bands of aquomet Mb ( imidazole complex ( ), and the difference spectrum ( ) in 0.1 M Tris buffer at pH 7.0 and 20°C. The spectrum of the imidazole complex was recorded 120 min after adding 3.0 mM of imidazole. (C) Soret absorption transition of Co(III) Mb after addition of 17.8 mM imidazole in 0.1 M Tris buffer at pH 7.0 and 20°C. (D) Time course of the 418 nm peak in (C).
Oxygen binding to Co(II) Mb The coordination structures of oxy and deoxy Co(II) Mb are identical to those of the corresponding Fe(II) Mb derivatives (15). We have measured the oxygen affinities of the Co(II) Mbs bearing several hemes (10). The P50 values for oxygen of proto-,
meso-, and deuteroheme Co(II) Mbs, taken from our previous report (10), were 50.0, 50.0, and 20.0 mm Hg, respectively, at 23°C (Fig. 2). However, the P50 values of Fe(II) Mbs containing proto(1.0 mm Hg), meso- (0.54 mm Hg), or deuteroheme (0.21 mm Hg) at the same condition were much
TABLE 1. Equilibrium and kinetic parameters of imidazole binding to Co(III) and Fe(III) Mb Materials Co(III) Mb Fe(III) Mb
kon (M/s)
koff (per second)
(4.4 ± 0.6) × 10 2.8 × 102
–2
(9.1 ± 0.8) × 10 4.5
–6
K (per M)
Reference
(4.8 × 1.1) × 10 62
2
This work (17)
Condition, pH 7.0 and 20°C for Co(III) Mb and pH 7.1 and 21–23°C for Fe(III) Mb.
FIG. 2. Structure of the prosthetic groups in Co(II) Mb. The values of P50 for O2 at 23°C are from our previous results (10).
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smaller (10). Thus, Co(II) Mbs exhibit lower oxygen affinity than Fe(II) Mbs. Generally, the P50 values in Co(II) Mbs were nearly comparable with or larger than the P50 = 10 mm Hg of Fe(II) Hb (10) and P50 = 32 mm Hg of red blood cells (6). The small P50 in deuteroheme Co(II) Mb among the three Mbs in Fig. 2 was ascribed to the decreased heme–globin interactions due to smaller bulk of the hydrogen atoms at the 2,4-positions of porphyrin (10). DISCUSSION Characteristic reactivity of Co(III) Mb The low reactivity of Co(III) Hb with CN- has been ascribed to an internal hemichrome structure. The same structural analogy seems to have been applied to Co(III) Mb. Our analysis, however, supported an aquomet structure for Co(III) Mb (15), and further suggested that the magnitude of hydrogen bonding between H2O and the distal histidine in Co(III) Mb is almost identical to that in aquomet Fe(III) Mb (11). We further found that various weak ligands coordinate to Co(III) Mb with rather high affinities. It is notable in Table 1 that the imidazole affinity to Co(III) Mb is moderately high, and that the association constant kon of imidazole is much smaller than that of Fe(III) Mb (17). The same holds for the kon and koff of the ligand in N3−, CN−, SCN−, and pyridine (11). The small kon constant observed for imidazole (Table 1) may be explained by the distinct electronic charge of Co(III) ion with six 3d-electrons, which is one electron larger than the Fe(III) ion with five 3d-electrons. The larger negative charge on the Co(III) ion could retard association of the unshared electron pair on imidazole. In addition, the Co(III)-N(imidazole) bond is shorter than the Fe(III)-N(imidazole) bond. For instance, Co(III)´˚ ) (18) is slightly comN(imidazole) bond (1.927 A pressed in comparison with the Fe(III)-N(imidazole) ´˚ ) (19) in the bis-imidazole adducts of bond (1.977 A metallo porphyrins. Interestingly, Nakamura demonstrated from the NMR analysis that the slightly shorter Co(III)-N(imidazole) bond in the Co(III) heme dramatically (approx. 1/105 fold) lowered the dissociation rate of the coordinating imidazole (20). Thus, the small koff rate in Co(III) Mb in Table 1 is reasonably explained in terms of the stronger Co(III)-imidazole bond than the corresponding bond in Fe(III) Mb. Utility of reduced Co(II) Mb as an Mb-based oxygen carrier The X-ray analysis of deoxy Co(II) Mb revealed that the Co(II) ion is five-coordinate (15). One Artif Organs, Vol. 38, No. 8, 2014
notable feature of Co(II) Mb, relative to Fe(II) Mb, is the much lower oxygen affinity. The P50 values of Co(II) Mbs bearing proto-, meso-, and deuterohemes are 50 to 100-fold larger than those of Fe(II) Mbs (Fig. 2). Much lowered oxygen affinities of Co(II) Mbs indicate that Fe(II) Mb is converted from the reservoir to carrier for oxygen after the metal substitution. With the physiological partial oxygen pressures in arterial (100 mm Hg) and venous (40 mm Hg) blood, we calculated the high transporting ability of oxygen in Co(II) Mbs with proto-, meso-, and deuteroheme to be 23, 23, and 50%, respectively (Fig. 2). It is well known that nitric oxide (NO) is a physiologic messenger molecule, and it has a high affinity to Hb. Possible NO capture by Co(II) Mb may induce vasoconstriction and increase blood pressure in the treated animals. The NO affinity of Co(II) Mb is the matter of significance. Rapson et al. reported the NO dissociation constants of 32 μM and 10 μM for Fe(II) Mb and Co(II) Mb, respectively (21), implying that the two Mbs have comparable NO affinities. A resonance Raman spectroscopic study indicated that the Co(II)-NO linkage is weaker than the Fe(II)-NO bond, owing to the steric constraint in the Co(II)-NO (22). The weaker Co(II)-NO bond may suggest a reduced NO affinity in Co(II) Mb; however, Co(II) Mb and Fe(II) Mb exhibit similar NO affinities (21). The similar NO affinities, in turn, indicate that Co(II) Mb has a larger kon rate of NO binding than Fe(II) Mb. This is consistent with the observation in ligand kinetics that Co(II) Mb (30 μM/s) has a larger kon rate than Fe(II) Mb (20 μM/s) (23). It is now widely accepted that carbon monoxide (CO) acts as a second messenger next to NO. Physiologic doses of CO, produced upon heme degradation by heme oxygenase, protect cells through potential anti-inflammatory, antiproliferative, or anti-apoptotic effects. Wang and coworkers recently demonstrated that CO is therapeutic in ischemiareperfusion brain injury (24). CO does not bind to Co(II) Mb at all while it has a high affinity to Fe(II) Mb. The lack of CO binding ability in Co(II) Mb is notable as it does not remove the physiological CO. CONCLUSION In summary, the present work provides an overview on the reactivity of cobalt-bearing myoglobin in the Co(III) and Co(II) states. Co(III) Mb exhibits characteristic spectroscopic and ligand-binding profiles despite that it has a common coordination structure with aquomet Fe(III) Mb. Co(II) Mb, however,
THOUGHTS AND PROGRESS has properties to show no CO affinity and low oxygen binding. The latter observation demonstrates a potential utility of Co(II)-substituted Mb as an oxygen carrier. Accordingly, Co(II) Mb is an attractive material to develop the Mb-based artificial oxygen carriers. Acknowledgments: This work was supported by Grants-in-Aid for the Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan, Nos. 23590040 (C) (to S.N.) and 24249086 (A) (to A.T.K.). We also thank the Yonetani Research Fund No. 2013 for the financial support to T.Y. REFERENCES 1. Kim HW, Greenburg AG. Artificial oxygen carriers as red blood cell substitutes: a selected review and current status. Artif Organs 2004;58:813–28. 2. Neya S, Suzuki M, Hoshino T. Novel controlling mechanism of oxygen affinity in myoglobin with isomeric porphyrins. Artif Organs 2009;33:189–93. 3. Neya S, Kawaguchi AT. Inherently distorted heme as a novel tool for myoglobin-based oxygen carrier. Artif Organs 2012; 36:220–3. 4. Neya S, Suzuki M, Hoshino T, et al. Molecular insight into intrinsic heme distortion in ligand binding in hemoprotein. Biochemistry 2010;49:5642–50. 5. Neya S, Funasaki N, Hori H, et al. Functional regulation of myoglobin by iron corrphycene. Chem Lett 1999;28:989–90. 6. Villela NR, Cabrales P, Tsai AG, Intaglietta M. Microcirculatory effects of changing blood hemoglobin oxygen affinity during hemorrhagic shock resuscitation in an experimental model. Shock 2009;31:645–52. 7. Dickinson LC. Metal replaced hemoproteins. J Chem Educ 1976;53:381–5. 8. Basolo F, Hoffman BM, Ibers JA. Synthetic oxygen carriers of biological interest. Acc Chem Res 1975;8:384–92.
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9. Chien JCW, Dickinson LC. Electron paramagnetic resonance of single crystal oxycobalt myoglobin and deoxy myoglobin. Proc Natl Acad Sci USA 1972;69:2783–7. 10. Yonetani T, Yamamoto H, Woodraw GV III. Studies on cobalt myoglobins and hemoglobins. J Biol Chem 1974;249: 682–90. 11. Neya S, Suzuki M, Hoshino T, Kawaguchi AT. Relaxation analysis of ligand binding to the myoglobin reconstituted with cobaltic heme. Inorg Chem 2013;52:7387–93. 12. Bernasconi CF. Relaxation Kinetics. New York: Academic Press, 1976;11–9. 13. Mortimer RG. Mathematics for Physical Chemistry. New York: Macmillan, 1981;266–309. 14. Rinsdale S, Cassatt JC, Steinhardt J. Acid denaturation studies on a cobalt(III) protoporphyrin-globin complex. J Biol Chem 1973;248:771–6. 15. Brucker EA, Olson JS, Phillips GN Jr, Dou Y, Ikeda-Saito M. High resolution crystal structure of the deoxy, oxy, and aquomet forms of cobalt myoglobin. J Biol Chem 1996;271:25419–22. 16. Brill AS, Williams RJP. The absorption spectra, magnetic moments and the binding of iron in some hemoproteins. Biochem J 1961;78:246–53. 17. Antonini E, Brunori M. Hemoglobin and Myoglobin in Their Reactions with Ligands. London: North-Holland Publishing, 1971;230. 18. Lauher JW, Ibers JA. Stereochemistry of cobalt porphyrins II. J Am Chem Soc 1974;96:4447–52. 19. Collins DM, Countryman R, Hoard JL. Stereochemistry of low spin iron porphyrins I. J Am Chem Soc 1972;94:2066–72. 20. Nakamura M. Effects of ortho-methyl substituents on the dissociation rate of imidazole ligand in tetraarylporphyrinatocobalt(III) complexes. Bull Chem Soc Jpn 1995;68:197–203. 21. Rapson TD, Dacres H, Trowell SC. Fluorescent nitric oxide detection using cobalt substituted myoglobin. Roy Soc Chem Adv 2014;4:10269–72. 22. Hu S. Resonance Raman spectroscopic studies of the nitric oxide adducts of cobaltous-reconstituted myoglobin and hemoglobin. Inorg Chem 1993;32:1081–5. 23. Quillin ML, Li T, Olson JS, et al. Structural and functional effects of apolar mutations of the distal valine in myoglobin. J Mol Biol 1995;245:416–36. 24. Wang B, Cao W, Biswal S, Doré S. Carbon monoxideactivated Nrf2 pathway leads to protecting against permanent focal cerebral ischemia. Stroke 2011;42:2605–10.
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