Formation of Compound I and Compound II Ferryl ...

Archives of Biochemistry and Biophysics Vol. 390, No. 2, June 15, pp. 304 –308, 2001 doi:10.1006/abbi.2001.2392, available online at http://www.idealibrary.com on

Formation of Compound I and Compound II Ferryl Species in the Reaction of Hemoglobin I from Lucina pectinata with Hydrogen Peroxide Walleska De Jesu´s-Bonilla, Jose´ E. Corte´s-Figueroa, Fernando A. Souto-Bachiller, Lolita Rodrı´guez, and Juan Lo´pez-Garriga 1 Chemistry Department, University of Puerto Rico, Mayaguëz Campus, P.O. Box 9019, Mayaguëz, Puerto Rico 00681-9019

Received February 27, 2001, and in revised form April 4, 2001; published online May 23, 2001

Hydrogen peroxide (H 2O 2) is a potent oxidant in vitro and in vivo that can reacts with heme proteins. It has been suggested that H 2O 2 is produced by the dismutation of the superoxide anion (O 2•⫺) (1, 2). Studies of heme peroxidases reacting with the H 2O 2 have revealed the formation of two main intermediary ferryl species (3– 6). One of these intermediates is the ferryl porphyrin cation radical (Fe IV ⫽ O Por 䡠 ⫹), namely,

compound I, which in turn may be reduced to its ferric state through the formation of heme ferryl (Fe IV ⫽ O Por) compound II. These ferryl species are present also in the reaction of myoglobin and hemoglobin with H 2O 2 (1, 4, 7, 8). Studies show that compound I and compound II are produced from a cyclic reaction in which the final products are the ferric heme and O 2•⫺ (9 –11). Recently, the rate constants in the microsecond time scale for the formation and reduction of compound I were measured by measuring the decay of the 417-nm band in sperm whale and horse heart ferric myoglobins (4). These results establish that compounds I and II are high valent iron derivatives that serve as essential intermediates in myoglobin peroxidative reactions. For instance, the reaction of the ferric myoglobin with mCPBA 2 forms myoglobin compound II without the formation of compound I (3). The reaction products of the ferric form of the H64A variant of myoglobin with m-CPBA show spectra characteristic of both compound I and compound II. This observation suggests that the distal histidine plays an important role in the stabilization of myoglobin compound I and that H64 substitution by amino acids like alanine, serine, and leucine accelerates the rate of reaction of m-CPBA and H 2O 2 with ferric myoglobin (4) and prolongs the lifetime of myoglobin compound I (3). Studies of the reaction of H 2O 2 with myoglobin variants (H64A, H64Q, L29F, V68F) (12, 13) suggested that the myoglobin variant H64Q/L29F produces a protein that removes the H 2O 2 more efficiently than the actual modified blood substitutes, preventing further oxidation of the remaining ferrous hemoglobin. Therefore, potential blood substi-

1 To whom correspondence should be addressed. Fax: (787) 2655476. E-mail: [email protected].

2 Abbreviations used: m-CPBA, m-choloroperbenzoic acid; HbI, Lucina pectinata hemoglobin I; Mb, myoglobin.

The formation of ferryl heme (Fe(IV) ⴝ O) species, i.e., compound I and compound II, has been identified as the main intermediates in heme protein peroxidative reactions. We report stopped-flow kinetic measurements which illustrate that the reaction of hemoglobin I (HbI) from Lucina pectinata with hydrogen peroxide produce ferryl intermediates compound I and compound II. Compound I appears relatively stable displaying an absorption at 648 nm. The rate constant value (kⴕ 2) for the conversion of compound I to compound II is 3.0 ⴛ 10 ⴚ2 s ⴚ1, more than 100 times smaller than that reported for myoglobin. The rate constant value for the oxidation of the ferric heme (kⴕ 12 ⴙ kⴕ 13) is 2.0 ⴛ 10 2 M ⴚ1 s ⴚ1. These values suggest an alternate route for the formation of compound II (by kⴕ 13) avoiding the step from compound I to compound II (kⴕ 2). In HbI from L. pectinata the stabilization of compound I is attribute to the unusual collection of amino acids residues (Q64, F29, F43, F68) in the heme pocket active site of the protein. © 2001 Academic Press Key Words: Lucina pectinata; hemoglobin I; hydrogen peroxide; ferryl.

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COMPOUNDS I AND II IN L. pectinata HEMOGLOBIN I

tutes can be produced by changing the heme pocket structure of hemoglobin and myoglobin (12, 14). The cDNA sequence and X-ray crystallographic studies of ferric hemoglobin I (HbI) from Lucina pectinata have shown that the Q64, F29, F43, and F68 amino acids residues are present in the heme pocket (15, 16). The distribution and structural organization of the residues in the region surrounding the heme pocket is analogous to H64Q/L29F/V68F and L29F/V68F myoglobin variants (12), the last one showing relatively high H 2O 2 decomposition turnover. Hemoglobin I binds and transfers H 2S forming a stable H 2S complex. This complex is hydrogen bonded to the oxygen in the carbonyl group of Q64 as shown by X-ray crystallography and resonance Raman experiments (15, 17). Metcyano-, carbon monoxy-, and oxy-HbI derivatives data from 1H NMR and resonance Raman predicted a model in which the iron of the heme group is tightly bonded to H96, but due to the absence of strong hydrogen bonding between the heme propionates and the nearby amino acids, the heme group is not firmly anchored. This heme group in HbI presents a rocking freedom that facilitates the binding between the heme and the incoming ligand (18). It is suggested also that the aromatic rings of the phenylalanine residues form an array in the HbI heme pocket, which acts as a multipole (19, 20) and is able to participate in electrostatic interactions that influence the ligand binding properties of HbI (17, 20). Here we present direct evidence for the stability of compounds I and II ferryl species upon reaction of ferric HbI from L. pectinata with H 2O 2 using the 648-nm band. We also report the reaction progress of ferric HbI species with H 2O 2. The reaction rates indicate that HbI has under ferric and ferro coordination a pseudoperoxidative and pseudocatalase activity toward removing H 2O 2. MATERIALS AND METHODS Hemoglobin I was isolated from the clam L. pectinata and purified according to the method described by Kraus and Wittenberg (21) and Navarro et al. (20). The ferric HbI was obtained from HbI-O 2 in phosphate buffer (pH 7.5) by adding potassium ferricyanide, whose excess was removed by ultrafiltration. The solutions of ferric HbI/ 30% H 2O 2 were prepared by mixing 900 ␮L of ferric HbI (17 ␮M) with equal volumes of various solutions of H 2O 2 (88 ␮M, 170 ␮M, 425 ␮M, 850 ␮M, and 17 mM). These solutions were used for the UV-Vis spectroscopic studies. The changes of the UV-Vis absorption spectrum from 350 to 700 nm were recorded at 1, 2, 6, 30, and 60 min after mixing. The absorption spectra were recorded also in a pH range from 5 to 10, to profile the stability of the HbI–H 2O 2 complex. The rate constants for the oxidation of ferric HbI with H 2O 2 were estimated using a modular Fluorolog-2 scanning spectrofluorometer system from SPEX with an absorbance/transmission assembly that allows measurement of transmitted radiation through a standard cuvette. An Olis (On-Line Instrument Systems Inc.) stopped-flow system equipped with a thermoelectrically cooled detector (190 – 860 nm) was used for the kinetic studies. The reaction of ferric HbI:H 2O 2 at ratios 1:5, 1:10, 1:25, 1:50, and 1:1000 were monitored by using the

SCHEME I

absorbance at 648 nm at 22°C over of 300 s, at 0.3-s intervals. A minimum of 300 data points were collected per experiment. The reaction of ferric HbI with H 2O 2 (Scheme I) is modeled by a scheme and mechanism similar to that suggested for myoglobin (4). The absorbance values at infinite time ( A ⬁ ) were determined by the Kezdy–Swibourne method (22, 23). The values of k 1obs and k 2obs were determined using a graphical procedure and a nonlinear least squares computation from data at 648 nm (24, 25). As the reaction has a physical property (absorbance) proportional to the concentration of the species, the equation that describes the reaction is obs

obs

共 A t ⫺ A ⬁ 兲 ⫽ ␣ e ⫺k 1 * t ⫹ ␤ e ⫺k 2 * t .

[1]

The kinetic constant values were estimated using similar approximations reported by Egawa and collaborators (4). The sum of the observable kinetic constants k 1obs and k 2obs in the overall reaction for ferric HbI reaction with H 2O 2 is defined as obs k obs 1 ⫹ k 2 ⫽ 共k⬘ 12 ⫹ k⬘ 21 ⫹ k⬘ 13 兲关H2O2兴 ⫹ k 23

[2]

where k⬘ 12 ⫹ k⬘ 21 ⫹ k⬘ 13 are bimolecular rate constants. The slope of the graph of k 1obs ⫹ k 2obs versus the H 2O 2 concentration in Eq. [2] gives the sum of k⬘ 12 , k⬘ 21 , and k⬘ 13 , and the intercept value is k 23 . The 648-nm band can be used to monitor the formation and autoreduction of HbI– compound I. The k 2obs is constant for each H 2O 2 concentration; thus it was assumed that k 2obs is independent of the H 2O 2 concentration. Therefore, the approximation given by Egawa and collaborators (4) was followed, where k⬘ 21 is negligible when compared with k⬘ 12 , k⬘ 21 , and k 23 . Then Eq. [2] is reduced to k obs 1 ⫽ 共k⬘ 12 ⫹ k⬘ 13 兲关H2O2兴 k

obs 2

⫽ k⬘ 2

[3] [4]

RESULTS AND DISCUSSIONS

Spectroscopic features of H 2O 2 with ferric HbI. The observed changes in the Soret band region provide clear evidence for the formation of HbI– compound I and II species. Figure 1 shows the overlaid spectra of ferric HbI during the reaction with H 2O 2 at 1:50 molar ratio. The L. pectinata ferric HbI hemoglobin has absorption maxima at 407, 502, and 633 nm. Upon application of an stoichiometric amount of H 2O 2, the Soret band shifts from 407 to 416 nm and its intensity decreases dramatically within the first second of H 2O 2 addition. After 1 min, a second shift from 416 to 419 nm was also observed. Upon addition of a large excess of H 2O 2, a shift from 407 to 419 nm was observed immediately as shown in Fig. 1. In the visible region (Fig. 1, inset), within the first minute after H 2O 2 addi-

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due to Q64 in the myoglobin variant H64Q (12). However, our study extends these observations by providing data on the kinetic behavior of compound I based on the 648-nm band. This allows to measure k⬘ 2 , the rate constant for the decay of compound I to compound II. Formation and decay rates of compound I and compound II. Figure 2 shows the changes in absorption at 648 nm using the stopped-flow instrument. At 1:5 molar ratio (ferric HbI:H 2O 2) the absorbance reaches a plateau approximately at 100 s, suggesting the accumulation of the intermediates that were produced in the pathways governed by k 12 and k 13 . This kinetics profile presents a gradual decay at 1:10 or larger molar ratios. Therefore, the formation of HbI– compound II or ferric HbI contributes to the observed behavior of the second section of the biphasic process. The Fig. 2 inset shows the absorption changes at 648 nm for 3 h. At longer times, the reaction shows almost the same behavior as at shorter times, suggesting a turnover of H 2O 2 consumption by hemoglobin I in a cyclic reaction pattern. The rate constant value for the oxidation of the ferric HbI (k⬘ 12 ⫹ k⬘ 13 ) taking into account Eq. [3] is 2.0 ⫻ 10 2 M ⫺1 s ⫺1. Studies of myoglobin variants (12) have FIG. 1. UV-Vis spectra monitoring the reaction of (A) ferric HbI ⬃ 17 ␮M with 900 ␮M H 2O 2 (1:50 ratio) (a) upon H 2O 2 addition and (b) after 30 min.

tion, the intensity of the ferric bands at 502 and 633 nm decreased to less than half the original value, most likely owing to heme degradation. New bands at 539, 569, and 580 nm and the compound I band at 648 nm appear. These spectral bands are similar to those reported by Alayash and collaborators (12) in the reaction of myoglobin variants with H 2O 2. Recent kinetic studies using the Soret band for native myoglobin (4) showed the formation of Mb-I on a 50-ms time scale. However, the band at 648 nm has not been previously discussed in the detail presented here. Studies of H64A, H64S, and H64L myoglobin variants with mCPBA (3) assign the 648-nm band to myoglobin compound I. The relatively long-lived formation of this 648-nm band in our study suggests the ability of HbI from L. pectinata to stabilize the ferryl compound I. This circumstance allows us to emphasize that the initial increase of the 648-nm band corresponds to the ferryl intermediate HbI– compound I, while the Soret band at 419 nm and the other bands at 539 and 580 nm involve the ferryl species HbI– compound II. The presence of longer lasting bands at 648, 419, and 580 nm suggests that the distal amino acids (Q64, F29, F43, F68) in the heme pocket of HbI stabilizes compound I and compound II species. This finding is in agreement with the stabilization of the H 2O 2 adduct

FIG. 2. Stopped-flow study of the reaction of ferric HbI with H 2O 2 monitored by the absorbance at 648 nm. The following molar ratios were used: (a) 1:5, (b) 1:10, (c) 1:25, (d) 1:50, (e) 1:1000. (Inset) UV-Vis time course mode of the reaction at the same molar ratios.

COMPOUNDS I AND II IN L. pectinata HEMOGLOBIN I

shown that the L29F/V68F Mb and H64Q/L29F Mb species have a rate constant value k⬘ 12 of 1.5 ⫻ 10 3 and 6.6 ⫻ 10 2 M ⫺1 s ⫺1, respectively. The corresponding value (k⬘ 12 ⫹ k⬘ 13 ) for sperm whale myoglobin (4) is 6.7 ⫻ 10 2 M ⫺1 s ⫺1. A large k⬘ 12 value implies a high H 2O 2 consumption rate in the peroxidative cycle. The rate constant value for the decay of HbI– compound I to compound II upon oxidation of HbI with H 2O 2, k⬘ 2 ⫽ k 2obs (Eq. [4]) is 3.0 ⫻ 10 ⫺2 s ⫺1 ⫾ 0.02, which is more than 100 times smaller than the value reported recently for the same constant of 4.0 s ⫺1 in myoglobin (4). The k⬘ 2 constant for L29F/V68F Mb and H64Q/L29F Mb variants is 7.9 ⫻ 10 ⫺3 and 1.0 ⫻ 10 ⫺2 s ⫺1, respectively. An important aspect is that compound I decay is the limiting step toward the formation of compound II ferryl species, which in turn forms the ferric HbI species. The low k⬘ 2 value for HbI suggests that the presence of H 2O 2 contributes to the stability of the heme ferric state. Since HbI is an unique hemoglobin that carries out its function in the heme ferric moiety, the result of the higher autoreduction constant implies that the binding of the H 2S ligand is facilitated, thus promoting the stability of the ferric H 2S complex. The results also indicate that at lower H 2O 2 concentrations the ferryl species predominates in the reaction, whereas when H 2O 2 is in excess, the autoreduction of HbI– compound I is affected by both the reduction of compound II to the ferric HbI and the H 2O 2 concentration. Roles of the distal amino acids in the reaction with H 2O 2. A further matter in this discussion is the importance of the heme pocket amino acids. Previous reports (3, 4, 12) showed that changing the distal histidine makes a myoglobin more resistant to the H 2O 2 damage, suggesting also that this residue is important in compound I stabilization. However, a clear description of the orientation of the amide components of the glutamine with respect to the ligand binding site is lacking in the literature. Crystallographic studies of ferric HbI with H 2O and H 2S suggested that the carbonyl group is pointing toward the iron, whereas studies with CN ⫺ suggested that the amino group is pointing toward the iron. Regarding this, HbI results confirm that the glutamine (Q64) and phenylalanines (F29, F48, F63) that surround the heme pocket help to stabilize the intermediate compound I and possibly compound II ferryl species. The pH results (data not shown) indicate a slight formation of the ferryl intermediate at basic pH and more favorable formation at neutral pH. The results suggest that the oxygen of the carbonyl group in Q64, which is oriented toward the heme iron, donates the electrons to form an initial hydrogen bond with the entering H 2O 2 molecule. In HbI this unique hydrogen bonding configuration promotes H 2O 2 binding by the carbonyl group of distal

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FIG. 3. Intermediate in the formation of HbI– compound I and compound II. The H–O bond in the H 2O 2 moiety bound to the heme iron is weakened by the hydrogen bond formation with the oxygen of the carbonyl group in Q64.

glutamine (Q64). Therefore, the Q64 stabilizes the complex more than the H64 counterpart explaining the long-lived 648-nm band. Figure 3 is a schematic representation of our model for the formation of the HbI– compound I and II precursor. The phenylalanines that surround the heme pocket (F29, F43, F68) create high electrostatic interactions (multipoles) (19), which stabilize the incoming ligand (17, 18, 20) and also contribute to the ferryl precursor formation. While the electrostatic stabilization toward the hydrogen bonding (Fig. 3a) and the Fe–O bond formation (Fig. 3b) are the same for all heme proteins with Q64 substitution with the same orientation, the electrostatic stabilization toward the other end of H 2O 2 should be a function of the particular heme pocket amino acids. In conclusion, the distal portion of heme pocket of L. pectinata HbI hemoglobin has three phenylalanines and a glutamine that make this hemoglobin unique. UV-Vis spectra indicate that HbI reacts with H 2O 2 at low and high peroxide concentrations. At higher peroxide concentrations, the reaction shows heme degradation in the final spectra. In this study, the 648- and the 419-nm bands are assigned to the formation of HbI– compound I and HbI– compound II ferryl species, respectively. L. pectinata HbI has the ability to stabilize, through its unusual heme pocket configuration, the ferryl compound I relative to that of myoglobin. The reactions of HbI with H 2O 2 suggest that HbI has the capability to consume up to 4 mol of H 2O 2 in the peroxidative cycle. Our results on the function of the Q64 emphasize the role played by the distal ligand residues on heterolytic O–O bond cleavage of H 2O 2. ACKNOWLEDGMENTS This project was supported, in part, by NSF (MCB-9974961) and NIH (MBRS/SCORE-SO6GM08103-27).

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REFERENCES 1. Fridovich, I. (1978) Science 201, 875– 880. 2. Tajima, G., and Shikama, K. (1993) Int. J. Biochem. 25, 101–105. 3. Matsui, T., Ozaki, S., and Watanabe, Y. (1997) J. Biol. Chem. 272, 32735–32738. 4. Egawa, T., Shimada, H., and Ishimura, Y. (2000) J. Biol. Chem. 275, 34858 – 66. 5. Chouchane, S., Lippai, I., and Magliozzo, R. S. (2000) Biochemistry 39, 9975–9983. 6. Iffland, A., Tafelmeyer, P., Saudan, C., and Johnsson, K. (2000) Biochemistry 39, 10790 –10798. 7. Yusa, K., and Shikama, K. (1987) Biochem. J. 26, 6684 – 6688. 8. Giulivi, C., and Davies, K. J. A. (1990) J. Biol. Chem. 265, 19453–19460. 9. Whitburn, K. D. (1987) Arch. Biochem. Biophys. 253, 419 – 430. 10. Whitburn, K. D. (1988) Arch. Biochem. Biophys. 267, 614 – 622. 11. Nagababu, E., and Rifkind, J. M. (2000) Biochem. Biophys. Res. Commun. 273, 839 – 845. 12. Alayash, A. I., Ryan, B. A., Eich, R. F., Olson, J. S., and Cashon, R. E. (1999) J. Biol. Chem. 274, 2029 –2037. 13. Matsui, T., Ozaki, S., Liong, E., Phillips, G., and Watanabe, Y. (1999) J. Biol. Chem. 274, 2838 –2844.

14. Snyder, S., Welty, E., Walter, R., William, L., and Walder, J. (1987) Proc. Natl. Acad. Sci. USA 84, 7280 –7284. 15. Rizzi, M., Wittenberg, J. B., Coda, A., Fasano, M., Ascenzi, P., and Bolognesi, M. (1994) J. Mol. Biol. 258, 1–5. 16. Antommattei, F. M., Rosado, T., Cadilla, C., and Lo´pez-Garriga, J. (1999) J. Protein Chem. 18, 831– 836. 17. Cerda, J. F., Echevarıá, Y., Morales, E., and Lo´pez-Garriga, J. (1999) Biospectroscopy 5, 289 –301. 18. Cerda, J. F., Silfa, E., and Lo´pez-Garriga, J. (1998) J. Am. Chem. Soc. 120, 9312–9317. 19. Thomas, K. A., Smith, G. M., Thomas, T. B., and Feldmann, R. J. (1982) Proc. Natl. Acad. Sci. USA 79, 4843– 4847. 20. Navarro, A. M., Maldonado, M., Gonza´lez-Lagoa, J., Lo´pezMejıá, R., Lo´pez Garriga, J., and Coloń, J. (1996) Inorg. Chim. Acta 243, 161–166. 21. Kraus, D. W., and Wittenberg, J. B. (1990) J. Biol. Chem. 265, 16043–16053. 22. Kezdy, F. J., Kaj, J., and Bruylants, A. (1958) Bull. Soc. Chim. Belges 67, 687. 23. Swibourne, E. S. J. (1960) J. Chem. Soc. 237. 24. Espenson, J. H. (1995) Chemical Kinetics and Reaction Mechanisms, pp. 25–17, 70 –76, McGraw–Hill, New York. 25. Corte´s-Figueroa, J. E., and Moore, D. A. (1999) J. Chem. Ed. 76, 635– 638.