Shokhireva Tatiana K, Shokhirev Nikolai V, Berry Robert E, Zhang Hongjun, Walker F Ann
Department of Chemistry, The University of Arizona, Tucson, AZ 85721-0041, USA.
J Biol Inorg Chem. 2008 Aug;13(6):941-59. doi: 10.1007/s00775-008-0381-8. Epub 2008 May 6.
The four major nitrophorins (NPs) of the adult blood-sucking insect Rhodnius prolixus have been reconstituted with the "symmetrical hemin" 2,4-dimethyldeuterohemin, and their NMR spectra have been investigated as the high-spin (S=5/2) aqua and low-spin (S=1/2) N-methylimidazole (NMeIm) and cyanide complexes. The NMeIm complexes allow assignment of the high-spin hemin resonances by saturation transfer difference spectroscopy. The cyanide complexes were investigated as paramagnetic analogues of the NO complexes. It is shown that the hemin ring is highly distorted from planarity, much more so for NP2 than for NP1 and NP4 (with ruffling being the major distortion mode), for both high- and low-spin forms. For the cyanide complexes, the conformation of the distorted ring changes on the NMR timescale to yield chemical exchange (exchange spectroscopy, EXSY) cross peaks for NP1sym(CN), NP3sym(CN) and NP4sym(CN) but not for NP2sym(CN). These changes in nonplanar conformation are visualized as a "rolling" of the ruffled macrocycle ridges through some number of degrees, the lowest-energy ruffling mode. This probably occurs in response to slow protein dynamics that cause the I120 and L132 side chains in the distal heme pocket to move in opposite directions (up and away vs. down and toward the hemin ring). This in turn changes the out-of-plane displacements of the 2M and 3M of the symmetrical hemin on the NMR timescale. Two other types of dynamics, i.e., changes in heme seating and NMeIm rotation, are also observed. The highly distorted heme and the dynamics it causes are unique to the NPs and a few other heme proteins with highly distorted macrocycles.
成年吸血昆虫红带锥蝽的四种主要硝基亚铁血红素蛋白（NPs）已用“对称血红素”2,4 - 二甲基氘代血红素进行了重构，并对其作为高自旋（S = 5/2）水合和低自旋（S = 1/2）N - 甲基咪唑（NMeIm）及氰化物配合物的核磁共振谱进行了研究。NMeIm配合物可通过饱和转移差光谱法确定高自旋血红素的共振峰。氰化物配合物作为NO配合物的顺磁类似物进行了研究。结果表明，无论高自旋还是低自旋形式，血红素环都严重偏离平面，NP2的这种情况比NP1和NP4更甚（褶皱是主要的畸变模式）。对于氰化物配合物，在核磁共振时间尺度上，畸变环的构象会发生变化，从而在NP1sym(CN)、NP3sym(CN)和NP4sym(CN)中产生化学交换（交换光谱法，EXSY）交叉峰，而NP2sym(CN)则没有。这种非平面构象的变化表现为褶皱大环脊通过一定角度的“滚动”，这是能量最低的褶皱模式。这可能是由于缓慢的蛋白质动力学导致远端血红素口袋中的I120和L132侧链向相反方向移动（向上和远离与向下和朝向血红素环）。这反过来又在核磁共振时间尺度上改变了对称血红素2M和3M的平面外位移。还观察到另外两种动力学类型，即血红素定位的变化和NMeIm的旋转。高度畸变的血红素及其引起的动力学是NP以及其他一些具有高度畸变大环的血红素蛋白所特有的。
