Abstract
A combination of one- and two-dimensional NMR experiments has been used to identify and spatially locate the heme pocket residues in the paramagnetic, low spin, met-cyano complex of elephant myoglobin. In addition to assigning resonances of the conserved residues, we have also assigned Gln64 (E7) and an aromatic ring designated PheA whose side chain is inserted into the heme pocket, as found earlier for elephant carbonmonoxy-myoglobin and oxy-myoglobin (Yu, L. P., La Mar, G. N., and Mizukami, H. (1990) Biochemistry 29, 2578-2585). The assigned conserved proximal side residues (Leu89(F4), Ala90(F5), His93(F8), His97(FG3), Ile99(FG5), Leu104(G5), Phe138(H15), and Tyr146(H23)) and conserved distal side residues (Phe43(CD1), Thr67(E10), Val68(E11), and Ala71(E14)) in elephant met-cyano-myoglobin are found to have orientations similar to those in sperm whale met-cyano-myoglobin. The observed dipolar connectivities and dipolar shift pattern for the substituted Gln64(E7) place the Gln in the heme pocket oriented toward the iron, as found for His64(E7). The conserved structural elements demand that the inserted PheA originate from the B-helix (i.e. Phe27 or Phe33). Dipolar contacts between the inserted PheA and the conserved residues Phe43(CD1), Val68(E11), Ile107(G8), and Gln64(E7), place PheA in the position occupied by the B10 residue in sperm whale myoglobin (Mb), with the larger size of the PheA side chain as compared to the replaced Leu being accommodated by the vacancy that occurs in sperm whale Mb. The paramagnetic induced relaxation places PheA in van der Waals contact with the bound ligand. Hence we conclude that the B10 position of elephant Mb is occupied by a Phe, and this substitution relative to sperm whale Mb is responsible for the low autoxidation rate and low reduction potential of elephant Mb. A reduced autoxidation rate has been reported for a sperm whale synthetic point mutant Leu29(B10) --> Phe (Carver, T. E., Brantley, R. E., Jr., Singleton, E. W., Arduini, R. M., Quillin, M. L., Phillips, G. N., and Olson, J. S. (1992) J. Biol. Chem. 267, 14443-14450). The published sequence of elephant Mb places B-helix Phe residues at position 27(B8) and 33(B14), but a Phe at neither of these positions can account for the observed NMR properties. Since a large proportion of the substitutions in elephant relative to sperm whale Mb, and some of the least conservative, occur in the B-helix, neither a structurally perturbed B-helix nor an error in the sequence can be discounted.
Highlights
Phe4'(CD1), Vala8(E11), Ile'0'(G8), and Glne4(E7), His(E7) + Gln point mutanthave shown that thesimple E7 place PheA in thpeosition occupied by the B10 residue substitution alone cannot account for the unusual Elephant myoglobin (El Mb) in sperm whale myoglobin (Mb), with the larger size properties (9)
The second observation is that methyl at -1.42 ppm is part of a three-spin system (Fig. 4), the two pocket residues of SW Mb that are conserved in El andthe NOESY cross-peakto8-CH3 (Fig. 5)identifies Mb (l),Phe33(B14) and Phe46(CD4), remain tboe identified
Resonance Assignments-The present results provide fur- proximal side of El Mb is minimally perturbed from that of therconfirmationthatmoderntwo-dimensional'HNMR
Summary
Steady-state NOEs from the broad labile proton at 19.2 ppm to both the Hisg3(F8) CoHs and the narrow labile proton peak at 13.51 ppm (not shown) confirm the earlier assignments of the 19.2 and 13.51 ppm peaks to the Hisg3(F8) ring NaH and peptide NH, respectively, as deduced earlier features of the proximal side (Fig. lA), and addressed solely from relaxation properties (23).The same dipolar conthe structural featuresof the distal side (Fig. 1B). This is con- 'H20 (Fig. 2D), the intensity of this peak in the difference firmed by a NOESY cross-peakbetween the Tyrlo3(G4) C,Hs trace at 6.5 ppm is less than one-third' that in 90% 'HzO, and another non-hyperfine shifted aromatic rinshgown to be establishing that the NOEis to thegeminal labile proton, i.e. a Phe (Phe'06(G7); seebelow).
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