Zhu Wenfeng, Li Yiming, Wang Jinling, Ortiz de Montellano Paul R, La Mar Gerd N
Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616, United States.
J Inorg Biochem. 2006 Jan;100(1):97-107. doi: 10.1016/j.jinorgbio.2005.08.010. Epub 2005 Dec 6.
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 alpha,gamma-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 approximately 1 A 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 degrees 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.
本文采用溶液质子核磁共振技术表明，无论是在中性pH条件下的高自旋铁原卟啉（PH）底物复合物，还是低自旋铁氰化物抑制的PH底物复合物，人血红素加氧酶-1（hHO）的233和265个残基重组构建体的活性位点电子结构和分子结构基本无法区分。此外，研究表明，在水配位的静止状态复合物中，PH围绕α,γ-中位轴的平衡取向异构比为1:1，但在氰化物抑制的复合物中变为4:1的平衡比，溶液中的次要异构体对应于晶体中发现的唯一一种。氰化物复合物中PH的取向偏好明显高于水合复合物，这可以通过对大鼠血红素加氧酶（rHO）相同的H2O和CN配位复合物的晶体学观察来解释，其中Fe-CN单元的空间倾斜导致PH向疏水内部移动约1 Å，并使乙烯基与HO基质的相互作用更强[M. Sugishima, H. Sakamoto, M. Noguchi, K. Fukugama, Biochemistry 42 (2003) 9898-9905]。氰化物抑制的PH复合物的1H NMR光谱最常用于确定HO复合物中PH的取向异构分布，也是最有用的。因此，为了得出与血红素的异构体位置和可变的异构体胆绿素产物相关的结论，必须考虑样品制备后光谱的时间进程。PH的天然取向异构导致光谱拥挤，这促使人们使用一种合成的、具有双重对称性的底物2,4-二甲基氘代血红素（DMDH）。虽然未配位残基的超精细位移模式非常相似，并且与替代底物的分子结构在很大程度上保守一致，但由主要磁轴方向确定的Fe-CN向量向蛋白质内部的空间倾斜，DMDH比PH小2度，这可以通过当庞大的乙烯基被甲基取代时底物在氰化物复合物中进一步向疏水内部移动来解释。
