Solution NMR study of environmental effects on substrate seating in human heme oxygenase: influence of polypeptide truncation, substrate modification and axial ligand.
Solution NMR study of environmental effects on substrate seating in human heme oxygenase: influence of polypeptide truncation, substrate modification and axial ligand.
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环境对人血红素加氧酶底物定位影响的溶液核磁共振研究：多肽截短、底物修饰和轴向配体的影响。
DOI：
10.1016/j.jinorgbio.2005.08.010
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发表时间：
2006
影响因子：
3.9
通讯作者：
LaMar,GerdN
中科院分区：
文献类型：
--
作者：
Zhu,Wenfeng;Li,Yiming;Wang,Jinling;OrtizdeMontellano,PaulR;LaMar,GerdN
溶液质子核磁共振已经被用来表明，无论是在中性pH下的高自旋铁，原氯化血红素(PH)底物络合物，还是低自旋铁，氰化物抑制的PH底物络合物，人血红素加氧酶-1，HHO的233个和265个残基的重组结构的活性中心电子和分子结构基本上是无法区分的。此外，还表明，在水连接的静止态配合物中，α，γ-MESO轴上的平衡PH取向异构体为1：1，而作为氰化物抑制的配合物，其平衡比例变为4：1，溶液中的少量物种与晶体中唯一的物种相对应。通过对大鼠血红素加氧酶(RHO)的相同H2O和CN连接的络合物的结晶学观察，合理地解释了在氰化物中引入显著的PH取向偏好是合理的，其中Fe-CN单元的空间倾斜导致PH进入疏水内部的∼1Å转变，以及乙烯基与Ho基质更强的相互作用[M.Sugishima，H.Sakamoto，M.Noguchi，K.Fukugama，BioChemical 42(2003)9898-9905]。氰化物抑制的PH络合物的1H核磁共振谱是最常用的，也是最有用的，用于确定PH的取向异构体在Ho络合物中的分布。因此，必须考虑样品制备后光谱的时间进程，以便得出结论，将血红素的异构体位置与不同的异构体胆绿素产物联系起来。PH的自然取向异构性导致光谱拥堵，这促使人们使用合成的双重对称底物2，4-二甲基亚铁血红素，DMDH。虽然非连接残基的超精细位移模式非常相似，并且与替代底物的高度保守的分子结构相一致，但Fe-CN载体向蛋白质内部的空间倾斜由主磁轴的取向决定，DMDH的空间倾斜比PH小2°，并且当大的乙烯基被甲基取代时，底物进一步进入氰化物复合体的疏水内部，从而使其合理化。
Solution proton NMR has been used here to show that, as either the high-spin ferric, protohemin (PH) substrate complex at neutral pH, or the low-spin ferric, cyanide-inhibited PH substrate complex, the active site electronic and molecular structure of the 233- and 265-residue recombinant constructs of human heme oxygenase-1, hHO, are essentially indistinguishable. It is shown, moreover, that the equilibrium PH orientational isomerism about the α,γ-meso axis is 1:1 in the water-ligated, resting-state complex, but changes to a 4:1 equilibrium ratio as the cyanide-inhibited complex, with the minor species in solution corresponding to the only one found in crystals. The introduction of significant PH orientational preference in the cyanide over the aquo complex is rationalized by the crystallographic observation for the same H2O and CN ligated complexes of rat heme oxygenase (rHO), where the steric tilt of the Fe–CN unit resulted in a ∼1Å transition of PH into the hydrophobic interior, and stronger interaction of the vinyls with the HO matrix [M. Sugishima, H. Sakamoto, M. Noguchi, K. Fukugama, Biochemistry 42 (2003) 9898–9905].1H NMR spectra of the cyanide-inhibited PH complex are the most used, and most useful, for determining the distribution of orientational isomerism for PH in complexes of HO. Hence, it is imperative that the time-course of the spectra after sample preparation be considered in order to reach conclusions that relate isomeric seating of the heme with variable isomeric biliverdin products. The natural orientational isomerism of PH leads to spectral congestion that has prompted the use of a synthetic, twofold symmetric substrate, 2,4-dimethyldeuterohemin, DMDH. While the hyperfine shift pattern for non-ligated residues are very similar and are consistent with largely conserved molecular structure with the alternate substrates, the steric tilt of the Fe–CN vector towards the protein interior, as determined by the orientation of the major magnetic axes, is 2° smaller for DMDH than PH, and is rationalized by the substrate translating even further into the hydrophobic interior in the cyanide complex when the bulky vinyl groups are replaced by methyl groups.