Tsai A L, Berka V, Chen P F, Palmer G
Division of Hematology, Department of Internal Medicine, University of Texas Medical School, Houston, Texas 77030, USA.
J Biol Chem. 1996 Dec 20;271(51):32563-71. doi: 10.1074/jbc.271.51.32563.
Electron paramagnetic resonance was used to characterize the heme structure of resting endothelial nitric-oxide synthase (eNOS), eNOS devoid of its myristoylation site (G2A mutant), and their heme complexes formed with 16 different ligands. Resting eNOS and the G2A mutant have a mixture of low spin and high spin P450-heme with widely different relaxation behavior and a stable flavin semiquinone radical identified by EPR as a neutral radical. This flavin radical showed efficient electron spin relaxation as a consequence of dipolar interaction with the heme center; P1/2 is independent of Ca2+-calmodulin and tetrahydrobiopterin. Seven of the 16 ligands led to the formation of low spin heme complexes. In order of increasing rhombicity they are pyrimidine, pyridine, thiazole, L-lysine, cyanide, imidazole, and 4-methylimidazole. These seven low spin eNOS complexes fell in a region between the P and O zones on the "truth diagram" originally derived by Blumberg and Peisach (Blumberg, W. E., and Peisach, J. (1971) in Probes and Structure and Function of Macromolecules and Membranes (Chance, B., Yonetani, T., and Mildvan, A. S., eds) Vol. 2, pp. 215-229, Academic Press, New York) and had significant overlap with complexes of chloroperoxidase. A re-definition of the P and O zones is proposed. As eNOS and chloroperoxidase lie closer than do eNOS and P450cam on the truth diagram, it implies that the distal heme environment in eNOS resembles chloroperoxidase more than P450cam. In contrast, 4-ethylpyridine, 4-methylpyrimidine, acetylguanidine, ethylguanidine, 2-aminothiazole, 2amino-4,5-dimethylthiazole, L-histidine, and 7-nitroindazole resulted in high spin heme complexes of eNOS, similar to that observed with L-arginine. This contrasting EPR behavior caused by families of ligands such as imidazole/L-histidine or thiazole/2-aminothiazole confirms the conclusion derived from parallel optical and kinetic studies. The ligands resulting in the low spin complexes bind directly to the heme iron, while their cognate ligands induce the formation of high spin complexes by indirectly perturbing the heme structure and excluding the original axial heme ligand in the resting eNOS (V. Berka, P.-F. Chen, and A. -L. Tsai (1997) J. Biol. Chem. 272, in press). The difference in EPR spectra of these high spin eNOS complexes, although subtle, are different for different homologs.
利用电子顺磁共振对静息型内皮型一氧化氮合酶（eNOS）、缺失肉豆蔻酰化位点的eNOS（G2A突变体）及其与16种不同配体形成的血红素复合物的血红素结构进行了表征。静息型eNOS和G2A突变体具有低自旋和高自旋P450 - 血红素的混合物，其弛豫行为差异很大，并且通过电子顺磁共振鉴定出一种稳定的黄素半醌自由基为中性自由基。由于与血红素中心的偶极相互作用，这种黄素自由基表现出有效的电子自旋弛豫；P1/2与Ca2 + - 钙调蛋白和四氢生物蝶呤无关。16种配体中的7种导致形成低自旋血红素复合物。按照菱形度增加的顺序，它们是嘧啶、吡啶、噻唑、L - 赖氨酸、氰化物、咪唑和4 - 甲基咪唑。这七种低自旋eNOS复合物位于最初由Blumberg和Peisach推导的“真值图”上的P区和O区之间的区域（Blumberg, W. E., and Peisach, J. (1971) in Probes and Structure and Function of Macromolecules and Membranes (Chance, B., Yonetani, T., and Mildvan, A. S., eds) Vol. 2, pp. 215 - 229, Academic Press, New York），并且与氯过氧化物酶的复合物有显著重叠。提出了对P区和O区的重新定义。由于在真值图上eNOS和氯过氧化物酶比eNOS和P450cam更接近，这意味着eNOS中血红素的远端环境与氯过氧化物酶更相似，而不是P450cam。相反，4 - 乙基吡啶、4 - 甲基嘧啶、乙酰胍、乙基胍、2 - 氨基噻唑、2 -氨基-4,5 - 二甲基噻唑、L - 组氨酸和7 - 硝基吲唑导致了eNOS的高自旋血红素复合物，类似于用L - 精氨酸观察到的情况。由咪唑/L - 组氨酸或噻唑/2 - 氨基噻唑等配体家族引起的这种对比鲜明的电子顺磁共振行为证实了从平行的光学和动力学研究得出的结论。导致低自旋复合物的配体直接与血红素铁结合，而它们的同源配体通过间接扰动血红素结构并排除静息型eNOS中原来的轴向血红素配体来诱导高自旋复合物的形成（V. Berka, P.-F. Chen, and A. -L. Tsai (1997) J. Biol. Chem. 272, in press）。这些高自旋eNOS复合物的电子顺磁共振光谱差异虽然细微，但不同同系物之间是不同的。
