Implication for using heme methyl hyperfine shifts as indicators of heme seating as related to stereoselectivity in the catabolism of heme by heme oxygenase: in-plane heme versus axial his rotation.
Implication for using heme methyl hyperfine shifts as indicators of heme seating as related to stereoselectivity in the catabolism of heme by heme oxygenase: in-plane heme versus axial his rotation.
复制标题
使用血红素甲基超细位移作为血红素座位的指标的含义与血红素加氧酶分解代谢中的立体选择性相关：平面内血红素与轴向旋转。
DOI：
10.1021/bi7017333
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发表时间：
2008
期刊：
影响因子：
2.9
通讯作者：
LaMar,GerdN
中科院分区：
文献类型：
--
作者：
Ogura,Hiroshi;Evans,JohnP;deMontellano,PaulROrtiz;LaMar,GerdN
人血红素加氧酶的溶解的265个残基构建体的三重突变体K18 E/E29 K/R183 E-hHO已经显示在血红素切割中将野生型hHO的排他性α-区域选择性重定向为主要的β，δ-选择性（Wang，J.，埃文斯，J. P.，Ogura，H.，拉马尔湾N.，和Ortiz de蒙泰拉诺，P. R.（2006）Biochemistry 45，61 - 73）。对三重突变体的氰化物抑制的原血红素和2，4-二甲基次血红素复合物的底物和轴向His CβH以及底物-蛋白质接触的1H NMR超精细位移模式进行了详细分析，并与WT复合物的数据进行了比较。结果表明，突变体中两种底物的主要溶液异构体的蛋白质接触决定了相对于WT中的蛋白质接触，在平面内顺时针旋转约90°。然而，对基质甲基超精细位移模式的常规解释表明，基质旋转仅为150 °。这一矛盾的解决表明，轴向His 25咪唑环也逆时针旋转相对于蛋白质基质的突变体相对于在WT。轴向His 25 CβH超精细位移被证明是咪唑平面相对于蛋白质基质取向的独立探针。分析表明，血红素甲基超精细位移的模式不能单独使用，以确定在平面内的基板的取向，因为它涉及到血红素裂解的立体特异性，没有明确考虑的轴向组氨酸咪唑平面相对于蛋白质基质的取向。
The triple mutant of the solubilized, 265-residue construct of human heme oxygenase, K18E/E29K/R183E-hHO, has been shown to redirect the exclusive α-regioselectivity of wild-type hHO to primarily β,δ-selectivity in the cleavage of heme (Wang, J., Evans, J. P., Ogura, H., La Mar, G. N., and Ortiz de Montellano, P. R. (2006)Biochemistry45, 61−73). The1H NMR hyperfine shift pattern for the substrate and axial His CβH's and the substrate−protein contacts of the cyanide-inhibited protohemin and 2,4-dimethyldeuterohemin complexes of the triple mutant have been analyzed in detail and compared to data for the WT complex. It is shown that protein contacts for the major solution isomers for both substrates in the mutant dictate ∼90° in-plane clockwise rotation relative to that in the WT. The conventional interpretation of the pattern of substrate methyl hyperfine shifts, however, indicates substrate rotations of only ∼50°. This paradox is resolved by demonstrating that the axial His25 imidazole ring also rotates counterclockwise with respect to the protein matrix in the mutant relative to that in the WT. The axial His25 CβH hyperfine shifts are shown to serve as independent probes of the imidazole plane orientation relative to the protein matrix. The analysis indicates that the pattern of heme methyl hyperfine shifts cannot be used alone to determine the in-plane orientation of the substrate as it relates to the stereospecificity of heme cleavage, without explicit consideration of the orientation of the axial His imidazole plane relative to the protein matrix.