[18 Anaerobic Oxidations of Myoglobin and Hemoglobin by Spectroelectrochemistry

By Céline H. Taboy, Celia Bonaventura, and Alvin L. Crumbliss

Introduction

A study of the redox properties of the myoglobins (Mbs) and hemoglobins (Hbs) provides insights into heme protein electron transfer processes, including the influence of subunit-subunit interactions and cooperativity. Studying the changes in redox behavior of various Mbs and Hbs has also allowed us and others to obtain important information regarding the involvement of iron in the transport of O2, NO, and other small molecules.1-68 In general terms,

1 C. Bonaventura, S. Tesh, K. M. Faulkner, D. Kraiter, and A. L. Crumbliss, Biochemistry 37, 496 (1998).

2 E. Antonini, J. Wyman, M. Brunori, J. F. Taylor, A. Rossi-Fanelli, and A. Caputo, J. Biol. Chem. 239,907 (1964).

3 K. M. Faulkner, A. L. Crumbliss, and C. Bonaventura, Inorg. Chim. Acta 226, 187 (1994).

4 E. Antonini and M. Brunori, "Hemoglobin and Myoglobin in Their Reactions with Ligands." Elsevier, North-Holland, Amsterdam, 1971.

5 E. W. Kristensen, D. H. Igo, R. C. Elder, and W. R. Heineman, ,/. Electroanal. Chem. 309, 61 (1991).

6 M. Brunori, G. M. Glacometti, E. Antonini, and J. Wyman, J. Biol. Chem. 63, 139 (1972).

7 M. Brunori, U. Saggese, G. C. Rotitio, E. Antonini, and J. Wyman, Biochemistry 10, 1604 (1971).

8 F. Riggs and R. A. Walbach, J. Gen. Physiol. 39, 585 (1956).

9 M. Bolognesi, S. Onesti, G. Gatti, A. Coda, P. Ascenzi, and M. Brunori, J. Mol. Biol. 205, 529 (1989).

10 M. Brunori, A. Alisen, U. Saggese, E. Antonini, and J. Wyman, J. Biol. Chem. 243, 2950 (1968).

11 M. Perutz, "Science Is Not a Quiet Life." World Scientific Publishing, Singapore, 1997.

12 R. J. Kassner, Proc. Natl. Acad. Sci. U.S.A. 69, 2263 (1972).

13 A. G. Mauk and G. R. Moore, J. Biol. Inorg. Chem. 2, 119 (1997).

14 M. R. Gunner, E. Alexov, E. Torres, and S. Lipovaca, J. Biol. Inorg. Chem. 2, 126 (1997).

15 E. Lloyd, B. C. King, F. M. Hawkridge, and A. G. Maulk, Inorg. Chem. 37, 2888 (1998).

16 J. Baldwin and C. Chothia, /. Mol. Biol. 129, 175 (1979).

17 A. Bellelli and M. Brunori, Methods Enzymol. 232,56 (1994).

18 M. Brunori, Á. F. Cutruzzola, C. Savino, C. Travaglini-Allocatelli, B. Vallone, and Q. H. Gibson, Biophys. J. 76, 1259 (1999).

19 D. H. Doherty, M. P. Doyle, S. R. Curry, R. Vali, T. J. Fattor, J. S. Olson, and D. D. Lemon, Nat. Biotechnol. 16, 672 (1998).

20 R. F. Eich, T. Li, D. D. Lemon, D. H. Doherty, S. R. Curry, J. F. Aitken, A. J. Mathews, K. A. Johnson, R. D. Smith, G. J. Phillips, Jr., and J. S. Olson, Biochemistry 35, 6976 (1996).

21 K. Imai, "Allosteric Effects in Hemoglobin." Cambridge University Press, Cambridge, 1982.

22 J. Monod, J. Wyman, and J. P. Changeux, J. Mol. Biol. 12, 88 (1965).

23 C. Travaglini-Allocatelli, A. F. Cutruzzola, A. Brancaccio, B. Vallone, and M. Brunori, FEBSLett. 352,63 (1994).

important issues such as the number of electrons involved in the redox process, cooperative effects between redox centers and between allosteric sites and redox centers, specific formal or midpoint potentials (Eor £1/2, respectively), and the shift in E\a due to chemical processes coupled to the redox process or to diverse effectors can be probed during a controlled redox

24 B. Vallone, P. Vecchini, V. Cavalli, and M. Brunori, FEBS Lett. 324, 117 (1993).

25 Z. Otwinowsky and W. Minor, Methods Enzymol. 276, 307 (1996).

26 F. A. Rossi and E. Antonini, Arch. Biochem. Biophys. 77,478 (1958).

27 W. Zhang, Ä. F. Cutruzzola, C. Travaglini-Allocatelli, M. Brunori, and G. La Mar, Biophys. J. 73, 1019 (1997).

28 J. E. Cradock-Watson, Nature (London) 215, 630 (1967).

29 B. Giardina, O. Brix, M. E. Clementi, R. Scatena, B. Nicoletti, R. Cicchetti, G. Argentin, and S. G. Condo, Biochem. J. 266, 897 (1990).

30 F. A. Armstrong, J. Biol. Inorg. Chem. 2, 139 (1997).

31 L. D. Kwiatkowski, H. L. Hui, A. Wierzba, R. W. Noble, R. Y. Walder, E. S. Peterson, S. G. Sligar, and K. E. Sanders, Biochemistry 37, 4325 (1998).

32 Y. Wu, E. Y. T. Chien, S. G. Sligar, and G. N. La Mar, Biochemistry 37, 6979 (1998).

33 J. F. Christian, M. Unno, J. T. Sage, P. M. Champion, E. Chien, and S. G. Sligar, Biochemistry 36, 11198 (1997).

34 S. J. Smerdon, S. Krzywda, A. M. Brzozowski, G. J. Davies, A. J. Wilkinson, A. Brancaccio, F. Cutruzzola, C. T. Allocatelli, M. Brunori, T. Li, R. E. Brandley, Jr., T. E. Carver, R. F. Eich, E. Singelton, and J. S. Olson, Biochemistry 34, 8715 (1995).

35 C. Bonaventura, G. Godette, S. Tesh, D. E. Holm, J. Bonaventura, A. L. Crumbliss, L. L. Pearce, and J. Peterson, J. Biol. Chem. 274, 5499 (1999).

36 C. Bonaventura and J. Bonaventura, Hemoglobin 4, 275 (1980).

37 C. Poyart, E. Bursaux, A. Arnone, J. Bonaventura, and C. Bonaventura, J. Biol. Chem. 255, 9465 (1980).

38 E. Bucci, A. Salahuddin, J. Bonaventura, and C. Bonaventura, J. Biol. Chem. 253, 821 (1978).

39 G. Amiconi, C. Bonaventura, J. Bonaventura, and E. Antonini, Biochim. Biophys. Acta 495, 279 (1977).

40 F. Bossa, D. Barra, M. Coletta, F. Martini, A. Liverzani, R. Petruzzelli, J. Bonaventura, and M. Brunori, FEBS Lett. 64, 76 (1976).

41 E. Chiancone, J. E. Nome, S. Forsen, J. Bonaventura, M. Brunori, E. Antonini, and J. Wyman, Eur. J. Biochem. 55, 385 (1975).

42 C. Bonaventura, J. Bonaventura, G. Amiconi, L. Tentori, M. Brunori, and E. Antonini, J. Biol. Chem. 250, 6273 (1975).

43 J. Bonaventura, C. Bonaventura, G. Amiconi, L. Tentori, M. Brunori, and E. Antonini, J. Biol. Chem. 250, 6278 (1975).

44 J. Bonaventura, C. Bonaventura, M. Brunori, B. Giardina, E. Antonini, F. Bossa, and J. Wyman, J. Mol. Biol. 82, 499 (1974).

45 D. Barra, F. Bossa, J. Bonaventura, andM. Brunori, FEBS Lett. 35, 151 (1973).

46 M. Brunori, J. Bonaventura, C. Bonaventura, B. Giardina, F. Bossa, and E. Antonini, Mol. Cell. Biochem. 1, 189 (1973).

47 J. S. Kavanaugh, D. R. Chafin, A. Arnone, A. Mozzarelli, C. Rivetti, G. L. Rossi, L. D. Kwiatkowski, and R. W. Noble, J. Mol. Biol. 248, 136 (1995).

48 G. E. Borgstahl, P. H. Rogers, and A. Arnone, J. Mol. Biol. 236, 817 (1994).

49 C. Poyart, E. Bursaux, A. Arnone, J. Bonaventura, and C. Bonaventura, J. Biol. Chem. 255, 9465 (1980).

reaction.3'7,1213'32'35'69-83 The spectroelectrochemical technique applied to Hbs and Mbs presented in this chapter offers an opportunity to specifically investigate the cooperativity in electron transfer between the four iron(II/III) sites in the Hbs and to compare the degree of redox cooperativity with that found for binding. Comparison between oxygenation and anaerobic oxidation can provide important information regarding the mechanism of hemoglobin function as well as enhanced understanding of heme protein electron transfer reactions. The purpose of this chapter is to outline the spectroelectrochemical technique used to study these various issues.

50 S. O'Donnell, R. Mandaro, T. M. Schuster, and A. Arnone, J. Biol. Chem. 254, 12204 (1979).

51 A. Arnone and M. F. Perutz, Nature (London) 249, 34 (1974).

54 A. M. Nigen, J. M. Manning, and J. O. Alben, J. Biol. Chem. 255, 5525 (1980).

55 M. H. Garner, R. A. Bogardt, Jr., and F. R. Gurd, J. Biol. Chem. 250,4398 (1975).

561. M. Russu and C. Ho, Biochemistry 25, 1706 (1986).

57 M. Perella, L. Benazzi, M. Ripamonti, and L. Rossi-Bernardi, Biochemistry 33,10358 (1994).

58 E. Chiancone, J. E. Nome, S. Forsen, J. Bonaventura, M. Brunori, and E. Antonini, Biophys. Chem. 3, 56(1975).

59 S. Bettati, A. Mozzarelli, and M. F. Perutz, J. Mol. Biol. 281, 581 (1998).

60 M. F. Perutz, D. T. B. Shih, and D. Williamson, J. Biol. Chem. 239, 555 (1994).

61 M. F. Perutz, G. Fermi, C. Poyard, J. Pagnier, and J. Kister,,/. Mol. Biol. 233, 536 (1993).

62 E. Chiancone, J. E. Nome, S. Forsen, E. Antonini, and J. Wyman, J. Mol. Biol. 70, 675 (1972).

63 R. Benesch and R. E. Benesch, Biochem. Biophys. Res. Commun. 26, 162 (1967).

64 C. H. Tsai, T. J. Shen, N. T. Ho, and C. Ho, Biochemistry 38, 8751 (1999).

65 A. Desbois and R. Baneijee, J. Mol. Biol. 92,479 (1975).

66 C. Ho and I. M. Russu, Biochemistry 26, 6299 (1987).

67 C. Bonaventura and J. Bonaventura, in "Biochemical and Clinical Aspects of Hemoglobin Abnormalities" (W. S. Caughey, ed.), p. 647. Academic Press, New York, 1978.

68 M. L. Meckstroth, B. J. Norris, and W. R. Heineman, Bioelectrochem. Bioenerget. 8, 63 (1981).

69 K. M. Faulkner, A. L. Crumbliss, and C. Bonaventura, J. Biol. Chem. 270, 13604 (1995).

70 R. J. Kassner, J. Am. Chem. Soc. 95,2674 (1973).

71 G. R. Moore, G. W. Pettigrew, and N. K. Rogers, Proc. Natl. Acad. Sei. U.S.A. 83,4998 (1986).

72 A. K. Churg and A. Warshel, Biochemistry 25, 1675 (1986).

73 T. Pascher, J. P. Chesick, J. R. Wrinkler, and H. B. Gray, Science 271, 1558 (1996).

74 A. Warshel, A. Papazyan, and I. Muegge, J. Biol. Inorg. Chem. 2, 143 (1997).

75 E. Lloyd, D. P. Hildebrand, K. M. Tu, and A. G. Mauk, J. Am. Chem. Soc. 117, 6434 (1995).

77 L. D. Dickerson, A. Sauer-Masarwa, N. Herron, C. M. Fendick, and D. H. Busch, J. Am. Chem. Soc. 115, 3623 (1993).

78 C. Brunei, A. Bondon, and G. Simonneaux, J. Am. Chem. Soc. 116, 11827 (1994).

79 J.-M. Lopez-Castillo, A. Filali-Mouhim, N. E. Van Binh-Otten, and J.-P. Jay-Gerin, J. Am. Chem. Soc. 119, 1978(1997).

80 J. Feitelson and G. McLendon, Biochemistry 30, 5051 (1991).

81 H. Durliat and M. Comtat, Anal. Chem. 54, 856 (1982).

82 S. J. Dong, J. J. Niu, and T. M. Cotton, Methods Enzymol. 246, 701 (1995).

83 M. L. Fultz and R. Durst, Anal. Chim. Acta 140, 1 (1982).

Although there exists a large body of literature that describes the mechanisms associated with oxygen binding to Hbs and Mbs, relatively little information has been reported with respect to the mechanism associated with tuning of the redox potential (£1/2) of these proteins.2,41°'65 Information regarding the influence of the prosthetic group environment on the overall propensity of the protein to accept or deliver electrons has been shown to lead to a more fundamental understanding of both 02 binding and anaerobic oxidation.1'3'35'69'84'85 Although a few results are presented here to illustrate these points, we direct the reader to Taboy et a/.84,85 for detailed discussions.

Spectroelectrochemical Technique and Cell Design

A limited number of techniques are available for investigating the redox properties of proteins. One of these is based on spectroelectrochemistry, in which spectral changes are monitored as a function of an applied potential. In other words, if a spectral difference exists between the oxidized and reduced form of the protein, information regarding its thermodynamic ease of oxidation (i.e., its half-potential, £1/2) and the number of electrons being transferred can be obtained. A combination of three components must be specifically chosen for each protein studied: (1) an electrode material and configuration, (2) a mediator or electron shuttle that facilitates electron transfer between the electrode and the protein, and (3) a detection system that can probe the presence of the oxidized and/or reduced state of the protein.5'81'82'84'86-93

Electrode

Different electrode materials can be used to perform a spectroelectrochemical experiment. The specific surface is chosen as a function of the applied potential window required to access the oxidation-reduction of the particular protein studied. This information is readily available in electrochemistry monographs such as that by Bard and Faulkner.94 Different electrode materials provide different

84 C. H. Taboy, C. Bonaventura, and A. L. Crumbliss, Bioelectrochem. Bioenerget. 48, 79 (1999).

85 C. H. Taboy, K. M. Faulkner, D. Kraiter, C. Bonaventura, and A. L. Crumbliss, J. Biol. Chem. 275, 39048 (2000).

86 K. Ashley and S. Pons, Chem. Rev. 88,673 (1988).

87 J. D. Brewster and J. L. Anderson, Anal. Chem. 54, 2560 (1982).

88 J. A. Cooper and R. G. Compton, Electroanalysis 10, 141 (1998).

90 W. R. Heineman, F. M. Hawkridge, and H. N. Blount, Electroanalysis 13, 1 (1984).

91 J. J. Niu and S. J. Dong, Rev. Anal. Chem. 15, 1 (1996).

92 R. S. Robinson, C. W. McCurdy, and R. L. McCreery, Anal. Chem. 54, 2356 (1982).

93 L. R. Sharpe, W. R. Heineman, and R. C. Elder, Chem. Rev. 90, 705 (1990).

94 A. J. Bard and L. R. Faulkner, "Electrochemical Methods: Fundamentals and Applications," 2nd Ed. John Wiley & Sons, New York, 2001.

overpotentials for solvent breakdown and therefore determine the effective electrochemical window available for a particular solvent/buffer system. For instance, platinum and gold mesh electrodes have been used successfully to study heme proteins and the transferrins, respectively, in our laboratory. Gold allows for a more negative potential window before breakdown of the buffer/solvent. The electrode material, of course, needs to be configured so that monochromatic light can be used as an oxidation state probe.

Optically Transparent Thin-Layer Cell

The most direct method for probing a redox change in a spectrally active metalloprotein is to record its UV-visible (UV-Vis) spectrum as the prosthetic group is being reduced or oxidized. A set of these spectra is most readily obtained by monitoring the changes in absorbance associated with the metal-ligand electronic properties at various applied potentials. An optically transparent thin-layer electrode (OTTLE) can be used and has the advantages of combining a small sample volume with maxium electrode surface-solution contact. This combination decreases the time required for the system to come to equilibrium at each applied potential. The experiment is essentially based on a bulk electrolysis process in which the bulk solution is delineated in space by the thickness of the OTTLE, typically leading to an ~0.03-cm diffusion layer.

Spectroelectrochemical experiments may be carried out using a two- or three-electrode arrangement (working, reference, and auxiliary) in an OTTLE cell as illustrated in Fig. 1. The cell is constructed from a 1 x 2 cm piece of 52 mesh platinum or gold gauze placed between the inside wall of a 1-cm path length cuvette and a piece of silica or quartz glass held in place by a small Tygon spacer positioned so as not to interfere with the spectral measurement.3 The OTTLE assembly results in an optical path length of 0.025-0.040 cm, depending on the electrode material and mesh size. A platinum (or gold) wire connects the platinum (or gold) gauze working electrode by insertion through a septum covering the top of the cuvette. A Pasteur pipette salt bridge plugged at the bottom with an agar gel containing 0.2 M KC1 is then prepared so as to connect an Ag/AgCl reference microelectrode to the solution containing the working electrode. The salt bridge solution is usually composed of the buffer used for the preparation of the working solution with a minimum of 0.2 Mbackground electrolyte (e.g., KC1). The auxiliary electrode is a 2 x 50 mm platinum (or 1 x 50 mm gold) wire inserted into one of the corners of the cuvette, so as not to obstruct the light path, and separated from the working gauze electrode by the piece of silica (or quartz). The platinum wire is held in place by the Tygon spacer at the bottom of the cell and the septum on top and presses against the silica (or quartz) piece, thereby sandwiching the working electrode tightly between the cell window and the silica (or quartz) piece. The sample solution fills the thin-layer portion of the cell surrounding the working

0 0

Post a comment