Quillin M L, Arduini R M, Olson J S, Phillips G N
Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77251.
J Mol Biol. 1993 Nov 5;234(1):140-55. doi: 10.1006/jmbi.1993.1569.
The highly conserved distal histidine residue (His64) of sperm whale myoglobin modulates the affinity of ligands. In an effort to fully characterize the effects of mutating residue 64, we have determined the high-resolution crystal structures of the Gly64, Val64, Leu64, Thr64 and Gln64 mutants in several liganded forms. Metmyoglobins with hydrophobic substitutions at residue 64 (Val64 and Leu64) lack a water molecule at the sixth coordination position, while those with polar amino acid residues at this position (wild-type and Gln64) retain a covalently bound water molecule. In the Thr64 mutant, the bound water position is only partially occupied. In contrast, mutating the distal histidine residue to glycine does not cause loss of the coordinated water molecule, because the hydrogen bond from the imidazole side-chain is replaced by one from a well-ordered solvent water molecule. Differences in water structure around the distal pocket are apparent also in the structures of deoxymyoglobin mutants. The water molecule that is hydrogen-bonded to the N epsilon atom of histidine 64 in wild-type deoxymyoglobin is not found in any of the position 64 mutant structures that were determined. Comparison of the carbonmonoxy structures of wild-type, Gly64, Leu64 and Gln64 myoglobins in the P6 crystal form shows that the conformation of the Fe-C-O complex is nearly linear and is independent of the identity of the amino acid residue at position 64. However, the effect of CO binding on the conformation of residue 64 is striking. Superposition of deoxy and carbonmonoxy structures reveals significant displacements of the residue 64 side-chain in the wild-type and Gln64 myoglobins, but no displacement in the Leu64 mutant. These detailed structural studies provide key insights into the mechanisms of ligand binding and discrimination in myoglobin.
抹香鲸肌红蛋白高度保守的远端组氨酸残基（His64）调节配体的亲和力。为了全面表征64位残基突变的影响，我们测定了几种配体形式下Gly64、Val64、Leu64、Thr64和Gln64突变体的高分辨率晶体结构。64位残基具有疏水取代（Val64和Leu64）的高铁肌红蛋白在第六配位位置缺少一个水分子，而该位置具有极性氨基酸残基的高铁肌红蛋白（野生型和Gln64）则保留一个共价结合的水分子。在Thr64突变体中，结合水的位置仅被部分占据。相比之下，将远端组氨酸残基突变为甘氨酸不会导致配位水分子的丢失，因为咪唑侧链的氢键被一个有序排列的溶剂水分子的氢键所取代。在脱氧肌红蛋白突变体的结构中，远端口袋周围水结构的差异也很明显。在已测定的任何64位突变体结构中，均未发现野生型脱氧肌红蛋白中与组氨酸64的Nε原子形成氢键的水分子。对P6晶型的野生型、Gly64、Leu64和Gln64肌红蛋白的一氧化碳结构进行比较表明，Fe-C-O复合物的构象接近线性，且与64位氨基酸残基的身份无关。然而，CO结合对64位残基构象的影响却很显著。脱氧和一氧化碳结构的叠加显示，野生型和Gln64肌红蛋白中64位残基侧链有明显位移，但Leu64突变体中没有位移。这些详细的结构研究为肌红蛋白中配体结合和识别机制提供了关键见解。
