Ligand Binding Properties of Myoglobin Reconstituted with Iron Porphycene: Unusual O 2 Binding Selectivity against CO Binding 1 Takashi Matsuo, ²,‡ Hirohisa Dejima, ² Shun Hirota, § Dai Murata, ² Hideaki Sato, ²,‡ Takahiro Ikegami, ² Hiroshi Hori, | Yoshio Hisaeda, ² and Takashi Hayashi* ,²,‡ Contribution from the Department of Chemistry and Biochemistry, Graduate School of Engineering, Kyushu UniVersity, Fukuoka 812-8581, Japan, PRESTO, Japan Science and Technology Agency (JST), Department of Physical Chemistry, Kyoto Pharmaceutical UniVersity, Yamashina, Kyoto 607-8414, Japan, and DiVision of Bioengineering, Graduate School of Engineering Science, Osaka UniVersity, Toyonaka, Osaka 560-8531, Japan Received July 9, 2004; E-mail: thayatcm@mbox.nc.kyushu-u.ac.jp Abstract: Sperm whale myoglobin, an oxygen storage hemoprotein, was successfully reconstituted with the iron porphycene having two propionates, 2,7-diethyl-3,6,12,17-tetramethyl-13,16-bis(carboxyethyl)- porphycenatoiron. The physicochemical properties and ligand bindings of the reconstituted myoglobin were investigated. The ferric reconstituted myoglobin shows the remarkable stability against acid denaturation and only a low-spin characteristic in its EPR spectrum. The Fe(III)/Fe(II) redox potential (-190 mV vs NHE) determined by the spectroelectrochemical measurements was much lower than that of the wild- type. These results can be attributed to the strong coordination of His93 to the porphycene iron, which is induced by the nature of the porphycene ring symmetry. The O 2 affinity of the ferrous reconstituted myoglobin is 2600-fold higher than that of the wild-type, mainly due to the decrease in the O2 dissociation rate, whereas the CO affinity is not so significantly enhanced. As a result, the O2 affinity of the reconstituted myoglobin exceeds its CO affinity (M′ ) KCO/KO2 < 1). The ligand binding studies on H64A mutants support the fact that the slow O2 dissociation of the reconstituted myoglobin is primarily caused by the stabilization of the Fe-O2 σ-bonding. The IR spectra for the carbon monoxide (CO) complex of the reconstituted myoglobin suggest several structural and/or electrostatic conformations of the Fe-C-O bond, but this is not directly correlated with the CO dissociation rate. The high O2 affinity and the unique characteristics of the myoglobin with the iron porphycene indicate that reconstitution with a synthesized heme is a useful method not only to understand the physiological function of myoglobin but also to create a tailor-made function on the protein. Introduction Myoglobin (Mb) is a dioxygen (O 2 ) storage hemoprotein having one protoporphyrin IX iron complex (heme) 2 1 as a prosthetic group and plays the role of facilitating O 2 diffusion to mitochondria through the muscle tissue of mammals. 3 The heme in Mb is fixed by multiple noncovalent interactions: coordination of His93 to the centered iron, hydrogen bondings of heme propionates with a vicinity of amino acid residues, and hydrophobic contact between peripheral heme alkyl chains and nonpolar amino acid residues in the hemepocket. 4,5 O 2 can be reversibly bound on the ferrous heme-iron in Mb, and the hydrogen bonding between the bound O 2 and His64 at the distal site is observed by X-ray crystallography 6a,b and neutron diffraction. 6c The nature of the Fe-O-O bond has also been discussed based upon the results of resonance Raman, 7 infrared resonance (IR), 7,8 and Mo ¨ ssbauer spectroscopic measurements. 9 ² Kyushu University. ‡ PRESTO, JST. § Kyoto Pharmaceutical University. | Osaka University. (1) Abbreviations: Mb, myoglobin; rMb, reconstituted myoglobin; wt-Mb(1), sperm whale wild-type myoglobin; wt-rMb(2), sperm whale reconstituted myoglobin with porphycene 2; H64A, the mutant whose 64th amino acid residue is replaced with alanine; H64A-Mb(1), sperm whale H64A myoglobin; H64A-rMb(2), sperm whale reconstituted H64A myoglobin with porphycene 2; hh-Mb(1), horse heart native myoglobin; hh-rMb(2), horse heart reconstituted myoglobin with porphycene 2; Hb, hemoglobin. (2) Strictly speaking, the term “heme” stands for a ferrous porphyrin. As a matter of convenience, we will employ this term throughout this paper for an iron porphyrin in a protein matrix, regardless of its oxidation state. (3) Hemoglobin and Myoglobin in Their Reactions with Ligands; Antonini, E., Brunori, M., Eds.; North-Holland: Amsterdam, 1971. (4) (a) Takano, T. J. Mol. Biol. 1977, 110, 537-568. (b) Ostermann, A.; Tanaka, I.; Engler, N.; Niimura, F.; Parak, F. G. Biophys. Chem. 2002, 95, 183-193. (5) Hargrove, M. S.; Barrick, D.; Olson, J. S. Biochemistry 1996, 35, 11293- 11299. (6) (a) Phillips, S. E. J. Mol. Biol. 1980, 142, 531-554. (b) Vojte ˇchovsky ´, J.; Chu, K.; Berendzen, J.; Sweet, R. M.; Schlichting, I. Biophys. J. 1999, 77, 2153-2174. (c) Phillips, S. E.; Shoenborn, B. P. Nature 1981, 292, 81- 82. (7) (a) Barlow, C. H.; Maxwell, J. E.; Wallace, W. J.; Caughey, W. S. Biochem. Biophys. Res. Commun. 1973, 55, 91-96. (b) Maxwell, J. C.; Volpe, J. A.; Barlow, C. H.; Caughey, W. S. Biochem. Biophys. Res. Commun. 1974, 58, 166-171. (8) (a) Tsubaki, M.; Nagai, K.; Kitagawa, T. Biochemistry 1980, 19, 379- 385. (b) Desbois, A.; Lutz, M.; Banerjee, R. Biochemsitry 1979, 18, 1510- 1518. (c) Yu, N.-T.; Benko, B.; Kerr, E. A.; Gersonde, K. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 5106-5110. (d) Spiro, T. G. Iron Porphyrins; Lever, A. B. P., Gray, H. B., Eds.; Addison Wesley: Reading, MA, 1983; Part II, pp 89-159. (9) Parak, B. D. Z. Naturforsch., C 1978, 33, 488-494. Published on Web 11/18/2004 10.1021/ja045880m CCC: $27.50 © 2004 American Chemical Society J. AM. CHEM. SOC. 2004, 126, 16007-16017 9 16007