5568 J. Am. Chem. zyxwvut SOC. zyxwvut 1986, 108, 5568-5573 (Abbott Laboratories) for helpful discussions and communication of unpublished experimental details. We are also indebted to Dr. Richard L. Keene of The Upjohn Company, Infectious Diseases Research and to Prof. DeShong for gifts of authentic tirandamycin A, and to John Schaeck for contributions to the project. The crystal structure analyses were performed by Dr. F. J. Hollander, staff crystallographer at the UC-Berkeley X-ray Crystallographic Facility (CHEXRAY). Support for our research by a grant from the National Institutes of Health (No. GM-30759) is gratefully acknowledged. Registry No. (1)-1, zyxwvutsrq 85880-71-3; (1)-9, 103383-32-0; (&)-lo, 103421-37-0; (1)-12, 103383-33-1; (1)-13, 103421-38-1; (1)-13 (ethyl ester), 103368-33-8; (1)-14, 103368-35-0 (f)-15, 103368-36-1; (1)-16, 103368-37-2; (1)-16-ol (isomer I), 103368-58-7; (1)-16-ol (isomer 2), 103421-42-7; (1)-18, 103368-59-8; (1)-20, 103368-60-1; (1)-22 (isomer l), 103368-61-2; (1)-22 (isomer 2), 103421-43-8; (1)-23, 103368-62-3; (f)-23.Na, 103421-44-9; (1)-23 (acid chloride), 103368-63-4; 25, 103368-56-5; 25 (acid), 103368-64-5; (1)-26, 103368-65-6; (A)-27, 103368-66-7; (1)-30, 103368-38-3; 32, 103368-39-4; 33, 103368-40-7; 42-9; (1)-36a, 103368-43-0; (f)-36/3, 103421-39-2; (1)-37c~, 103368- 67-8; (A)-38, 103383-08-0; (1)-39, 103368-44-1; (1)-40, 103368-46-3; (1)-41, 103368-45-2; (1)-43, 103368-47-4; (1)-44uc~, 103368-48-5; (1)-34/3, 103368-31-6; (1)-34@ (TMS enol), 103368-32-7; 35, 103368- (1)-44a& 103421-40-5; (1)-45, 103368-41-8; (1)-45 (TBDMS ether), 103368-49-6; (1)-45 (bromosilyl derivative), 103368-50-9; (1)-48, 103368-51-0; (1)-48 (disilated), 103368-52-1; (1)-48 (desilated ketone), 103368-53-2; (1)-49, 103421-41-6; (1)-49 (silyl enol), 103368-54-3; 97859-87-5; (k)-(trans)-ethyl 2.3-epoxybutyrate, 82769-14-0; [3R*,4R1,5S*( IS*)]-3-hydroxy-4-methyl-5-( 1 -iodoethyl)dihydro-2- (3H)-furanone, 103368-34-9; propyne, 74-99-7; hexafluroacetone, 684- 16-2; methyl isopropenyl ketone, 814-78-8; ethyl vinyl ether, 109-92-2; 3-methoxy-3-methylbutyne, 13994-57-5; methyl propiolate, 922-67-8. Supplementary Material Available: Experimental details for the synthesis and characterization of the compounds depicted in Schemes I11 and IV, as well as compounds 37 and 38 (5 pages). Ordering information is given on any current masthead page. (1)-50, 103421-36-9; (1)-51, 103368-55-4; (1)-52, 103368-57-6; (1)-53, Influence of Propionate Side Chains on the Equilibrium Heme Orientation in Sperm Whale Myoglobin. Heme Resonance Assignments and Structure Determination by Nuclear Overhauser Effect Measurements Gerd N. La Mar,* S. Donald Emerson, Juliette T. J. Lecomte, Usha Pande, Kevin M. Smith, G. Wayne Craig, and Lisa A. Kebres Contribution from the Department of Chemistry, University zyxw of California, Davis, California 95616. Received February 28, 1986 Abstract: Sperm whale myoglobin was reconstituted with hemins methylated at the 2-, 4-, and 6- or 7-positions, and the corresponding metcyano complexes were studied by ‘H NMR spectroscopy. Nuclear Overhauser effects (NOES) were observed between heme methyls attached to the same pyrrole ring and between heme methyls adjacent to a common meso position. The relative magnitudes of these effects could be relied on to identify heme methyl resonances and are proposed to be used as a simple method to reach spectral assignments. Along with selected and characteristic dipolar contacts with clearly identified peripheral protein side chains, the inter-methyl NOE allows direct determination of the heme orientation in the holoprotein. Thus, it was found that the replacement of either propionate side chain with a methyl does not affect the nature of the thermodynamically preferred isomer and does not perturb the equilibrium proportion of the two species. The two alternate orientations of the heme in b-type hemo- proteins involve a 180’ rotation about the zyxwvutsrq a,y meso axis (A in Figure and have been shown to play key roles in the initial steps of the in vitro assembly from apoprotein and heme for my~globin,~ hemoglobin$ and cytochrome b5.5 In the former two proteins, preliminary evidence suggests that the in vivo assembly may proceed via similar pathway^.^,^ Moreover, not only does the initial step of the reaction between heme and apoprotein fail to distinguish between the two sides of the heme but both heme orientations remain populated to some degree at equilibrium, (1) La Mar, G. N.; Budd, D. L.; Viscio, D. B.; Smith, K. M.; Langry, K. (2) La Mar, G. N.; Davis, N. L.; Parish, D. W.; Smith, K. M. J. Mol. Bioi. (3) Jue, T.; Krishnamoorthi, R.; La Mar, G. N. J. Am. Chem. Soc. 1983, (4) La Mar, G. N.; Yamamoto, Y.; Jue, T.; Smith, K. M.; Pandey, R. K. Biochemisrry 1985, 24, 3826-3831. (5) La Mar, G. N.; Burns, P. D.; Jackson, T. J.; Smith, K. M.; Langry, K. C.; Stritmatter, P. J. Bioi. Chem. 1981, 256, 6075-6079. (6) Levy, M. J.; La Mar, G. N.; Jue, T.; Smith, K. M.; Pandey, R. K.; Smith, W.; Livingston, D. J.; Brown, W. D. J. Bioi. Chem. 1985, 260, C. Proc. Natl. Acad. Sci. USA. 1978, 75, 5755-5159. 1983, 168, 887-896. 105, 5701-5702. 13694-1 3698. leading to equilibrium heme rotational d i ~ o r d e r . ~ , ~ This equilib- rium disorder in both mammalian myoglobin and hemoglobin A involves only 10-15% of the “reversed” heme orientation as in the lower part of A in Figure zyxw 1. However, considerably larger degrees of disorder have been found in insect hemoglobin,’ fish myoglobin,6 and mammalian myoglobin reconstituted with chemically modified 2,4-~ubstituents.~ We have shown previously that the nature of the 2,4-substituents inflllences both the rate of heme reorientation and the position of the equilibrium between the two heme orientations.8 In the present study, we extend our investigation to explore the influence of the modification of the heme 6- or 7-propionate chains on equilibrium heme orientation in sperm whale Mb. Holoproteins of sperm whale Mb at apparent equilibrium were prepared for the two modified hemes, 7-(2-carboxyethyl)-l,3,5,6,8-penta- methyl-2,4-divinylhemin and 6-(2-~arboxyethyl)-l,3,5,7,8- pentamethyl-2,4-di~inylhemin,~ hereafter referred to as 6- (7) La Mar, G. N.; Smith, K. M.; Gersonde, K.; Sick, H.; Overkemp, M. (8) La Mar, G. N.; Toi, H.; Krishnamoorthi, R. J. Am. Chem. SOC. 1984, J. Bioi. Chem. 1980, 255, 266-270. 106, 6395-6401. 0002-7863/86/ 1508-5568$01.50/0 0 1986 American Chemical Society