La Mar G N, Thanabal V, Johnson R D, Smith K M, Parish D W
Department of Chemistry, University of California, Davis 95616.
J Biol Chem. 1989 Apr 5;264(10):5428-34.
High field deuterium NMR spectra have been recorded for various horseradish peroxidase complexes reconstituted with hemins possessing specific 2H labels. The line width of the 2H NMR signals of deuteroheme reconstituted-horseradish peroxidase (HRP) and its cyano complex for the immobilized skeletal 2-2H and 4-2H labels yield the overall protein rotational correlation time (22 ms at 55 degrees C), which is consistent with expectations based on molecular weight. Meso-2H4 labels yield broad (1.3 kHz) signals just upfield from the diamagnetic protein envelope for HRP, and in the central portion of the protein envelope for the CN- ligated resting state HRP. Meso-2H4-labeled mesohemin-reconstituted HRP exhibits a similar signal but shifted further upfield by approximately 10 ppm. The net upfield meso-H hyperfine shifts confirm a five-coordinate structure for resting state HRP. 2Ha resonances for essentially rotationally immobile vinyl groups were detected in both resting state HRP and CN- ligated resting state HRP. Heme methyl-2H-labeling yields relatively narrow lines (approximately 80 Hz) indicative of effective averaging of the quadrupolar relaxation by rapid methyl rotation. Thus the 2H line width of rapidly rotating methyls in hemoproteins can be used effectively to determine the overall protein tumbling rate. Preliminary 2H experiments in meso-2H4-labeled compound I do not support large pi spin density at these positions on the porphyrin cation radical, and argue for a a1u rather than a a2u orbital ground state.
已记录了用具有特定2H标记的血红素重构的各种辣根过氧化物酶复合物的高场氘核磁共振光谱。固定化骨架2-2H和4-2H标记的氘代血红素重构辣根过氧化物酶（HRP）及其氰基复合物的2H NMR信号的线宽给出了整体蛋白质旋转相关时间（55℃时为22毫秒），这与基于分子量的预期一致。中-2H4标记在HRP的抗磁性蛋白质包络线的正上方产生宽（1.3千赫兹）信号，而在CN连接的静止状态HRP的蛋白质包络线的中心部分产生该信号。中-2H4标记的中血红素重构的HRP表现出类似的信号，但进一步向上场移动了约10 ppm。净向上场中-H超精细位移证实了静止状态HRP的五配位结构。在静止状态HRP和CN连接的静止状态HRP中均检测到基本旋转不动的乙烯基的2Ha共振。血红素甲基-2H标记产生相对较窄的线（约80赫兹），表明通过快速甲基旋转有效地平均了四极弛豫。因此，血红蛋白中快速旋转甲基的2H线宽可有效地用于确定整体蛋白质翻滚速率。中-2H4标记的化合物I的初步2H实验不支持卟啉阳离子自由基上这些位置的大π自旋密度，并支持a1u而非a2u轨道基态。
