Vyas K, Rajarathnam K, Yu L P, Emerson S D, La Mar G N, Krishnamoorthi R, Mizukami H
Department of Chemistry, University of California, Davis 95616.
J Biol Chem. 1993 Jul 15;268(20):14826-35.
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.
一维和二维核磁共振实验相结合，已用于鉴定和在空间上定位非洲象肌红蛋白顺磁性、低自旋、高铁氰基复合物中的血红素口袋残基。除了归属保守残基的共振信号外，我们还归属了Gln64（E7）和一个名为PheA 的芳香环，其侧链插入到血红素口袋中，这与之前在非洲象碳氧肌红蛋白和氧合肌红蛋白中发现的情况相同（于立平、拉马尔、水神晃（1990年）《生物化学》29卷，2578 - 2585页）。所归属的非洲象高铁氰基肌红蛋白中保守近侧残基（Leu89（F4）、Ala90（F5）、His93（F8）、His97（FG3）、Ile99（FG5）、Leu104（G5）、Phe138（H15）和Tyr146（H23））以及保守远侧残基（Phe43（CD1）、Thr67（E10）、Val68（E11）和Ala71（E14）），发现其取向与抹香鲸高铁氰基肌红蛋白中的相似之处。观察到的取代Gln64（E7）的偶极连接性和偶极位移模式表明，Gln在血红素口袋中朝向铁的方向，这与His64（E7）情况相同。保守的结构元件要求插入的PheA源自B - 螺旋（即Phe27或Phe33）。插入的PheA与保守残基Phe43（CD1）、Val68（E11）、Ile107（G8）和Gln64（E7）之间的偶极接触将PheA置于抹香鲸肌红蛋白（Mb）中B10残基所占据的位置，与被取代的Leu相比PheA侧链更大的尺寸由抹香鲸Mb中出现的空位所容纳。顺磁诱导弛豫使PheA与结合的配体处于范德华接触。因此我们得出结论，非洲象Mb的B10位置被一个Phe占据，相对于抹香鲸Mb的这种取代是非洲象Mb自氧化速率低和还原电位低的原因。已报道抹香鲸的一个合成点突变Leu29（B10）→Phe自氧化速率降低（卡弗、布兰特利、小R.E.、辛格尔顿、R.M.阿尔杜伊尼、M.L.奎林、G.N.菲利普斯、J.S.奥尔森（1992年）《生物化学杂志》267卷，14443 - 14450页）。已发表的非洲象Mb序列在B - 螺旋位置27（B8）和33（B14）处有Phe残基，但这两个位置的Phe都无法解释观察到的核磁共振性质。由于相对于抹香鲸Mb，非洲象中很大一部分取代，并且是一些最不保守的取代，发生在B - 螺旋中，所以不能排除结构受到干扰的B - 螺旋或序列中的错误。
