698 1
J. Am. Chem. SOC.1994,116, 6981-6982
Functional Cavities in Proteins: A General Method for Proximal Ligand Substitution in Myoglobin
His 64
Gia D. DePillisJ Sean M.DecaturJ Doug Barrick,: and Steven G. Boxer'st
Phe 43
Received December 9, 1993
The amino acid ligands to the heme iron provide an important distinction between different classes of heme proteins. For example, the peroxidases and the oxygen carrier proteins myoglobin (Mb) and hemoglobin bear a proximal His, electrontransfer proteins such as cytochrome c have His, Met, and Lys ligands, and oxidases such as cytochrome P450 and catalase contain Cys and Tyr ligands, respectively. In contrast to chemical model systems, where a large range of ligands has been used to modulate heme function,' replacement of the proximal ligand in Mb has been limited in scope to the natural amino acids Cyszand Tyr.2~~ In order to understand the vast range of reactivitiesamong heme proteins, it would be ideal to possess the flexibility to vary the proximal ligand, while retaining the structure, recognition, specificity, and ease of manipulation provided by the protein matrix. We report a simple method to achieve this goal for myoglobin, with broader implications for protein engineering in generals4 When the proximal His residue 93 in sperm whale Mb is changed to Gly by site-directed mutagenesis and the protein is expressed in Escherichia coli5 using a medium containing imidazole (Im), a protein is isolated which has a molecular weight and electronic absorption spectrum similar to those of wild-type (WT) Mbe6 The high-resolution X-ray crystal structure of this protein, H93G(Im), illustrated schematicaliy in Figure 1,shows that Im occupies the cavity created by conversion of His93 to Gly and is coordinated to the heme iron.' As shown in Figure 2, replacement of proximal His93 by Im leads to significant changes Stanford University. t University of Oregon.
(1) Ji, L.-N.; Liu, M.; Hsieh, A.-K.; Hor, T. S.A. J . Mol. Catal. 1991, 70, 247-257. James, B. R. In The Porphyrins; Dolphin, D.. Ed.; Academic
Press: New York, 1978; Vol. V, pp 205-302. (2) Adachi, S.;Nagano, S.;Ishimori, K.; Watanabe, Y.; Morishima, I.; Egawa,T.;Kitagawa,T.;Makino,R. Biochemistry 1993,32,241-252. Adachi, S.; Nagano, S.;Watanabe, Y.; Ishimori, K.; Morishima, I. Biochem. Biophys. Res. Commun. 1991, 180, 138-144. (3) Egeberg,K. D.; Springer, B.A.; Martinis, S.A.;Sligar, S.G.; Morikis, D.; Champion, P. M. Biochemistry 1990, 29, 9783-9791. (4) Alternative methods include protein semisynthesis, which has been applied to cytochrome c (Wuttke, D. S.; Gray, H. B.; Fisher, S.L.; Imperiali, B. J . Am. Chem.Soc. 1993, I15,8455-8456. Wallace,C. J. A.;Clark-Lewis, I. J . Biol. Chem. 1992, 267, 3852-3861), and chemical aminoacylation of suppressortRNA complementaryto a nonsense codon introduced at thedesired site, followed by in uitro protein expression (Noren, C. J.; Anthony-Cahill, S.J.;Griffith, M.C.;Schultz,P.G.Science 1989,244,182-188). Thestrategy of substituting amino acid side chains with exogenous molecules has been applied to, e.&, aspartate amino transferase (Toney, M. D.; Kirsch, J. F. Science 1989,243,1485-1488); subtilisin (Carter, P.; Abrahmsen, L.; Wells, J. A. Biochemistry 1991, 30, 6142-6148); trypsin (Perona, J. J.; Hedstrom, L.;Wagner, R. L.; Rutter, W. J.; Craik, C. S.;Fletterick, R. J. Biochemistry 1994,33,3252-3259); rhodopsin (Zhukovsjy, E. A.; Robinson, P. R.; Oprian, D. D. Science 1991,252, 558-560); and azurin (den Blaauwen, T.; Canters, G. W. J . Am. Chem. Soc. 1993, 115, 1121-1129). ( 5 ) Springer, B. A.; Sligar, S.G. Proc. Null. Acad. Sci. U.S.A. 1987,84, 8961-8965. (6) Barrick, D. Biochemistry 1994, 33, 6546-6554. (7) Relevant differences in the structure of H93G(Im) compared to WT reported in ref 6 include (1) Fe-N bond length 0.3 A shorter; (2) imidazole lane rotated 36' relative to the His93 plane; (3) nonliganded Im N atom 0.2 closer to the hydroxyl roup of Ser92, adopting a favorable hydrogenbonding geometry; (4) 0.6 increase in the distance between Fe and the distal H20; and ( 5 ) Fe 0.2 A closer to the plane of the heme.
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Val 68 'C
Department of Chemistry, Stanford University Stanford, California 94305-5080 Institute for Molecular Biology University of Oregon, Eugene, Oregon 97403
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od Gly93 Figure 1. Schematic diagram of heme pocket residues in carbonmonoxymyoglobin mutant H93G(Im),6 illustrating the replacement of proximal His93 with free imidazole and further exchange with other small ligands L. 1 .o n
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