Matsuoka A, Stuehr D J, Olson J S, Clark P, Ikeda-Saito M
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106-4970.
J Biol Chem. 1994 Aug 12;269(32):20335-9.
Neuronal nitric oxide synthase (NOS) is a calmodulin-dependent, flavin-containing hemoprotein that forms NO from L-arginine, NADPH, and molecular oxygen. Calmodulin binding to NOS triggers reduction of its heme groups (Abu-Soud, H., and Stuehr, D.J. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 10769-10762), leading to NADPH oxidation and NO synthesis. We have examined how L-arginine and calmodulin control the ligand binding and electron acceptor properties of the NOS heme iron. In the absence of bound calmodulin, ferric NOS exhibited a Kd of 0.6 microM for L-arginine, as determined by the substrate-dependent shift in heme spin equilibrium toward a high spin state. L-Arginine binding reduced the affinity of the ferric NOS heme for cyanide by 8-fold. Carbon monoxide binding to substrate-free ferrous NOS occurred at a rate of 2 x 10(5) M-1 S-1; this rate was decreased 12-fold when L-arginine was bound. In contrast, bound calmodulin did not significantly affect cyanide or carbon monoxide binding to the NOS heme, nor did it alter NOS binding affinity for L-arginine. Anaerobic titration of a calmodulin-bound, L-arginine-free NOS with NADPH led to incomplete reduction of the heme iron; full reduction was achieved only in the presence of added L-arginine. Thus, our data suggest that L-arginine controls NOS heme iron reactivity in at least two ways: 1) it slows ligand interactions by binding in the distal pocket very near the heme and 2) it also appears to increase the reduction potential of the iron. In contrast, bound calmodulin does not alter the NOS affinity for L-arginine or heme ligands and may function solely as a switch that enables electrons to pass from the flavin domain onto the heme iron.
神经元型一氧化氮合酶（NOS）是一种钙调蛋白依赖性、含黄素的血红素蛋白，它利用L-精氨酸、NADPH和分子氧生成一氧化氮。钙调蛋白与NOS结合会触发其血红素基团的还原（阿布-苏德，H.，和斯图尔，D.J.（1993年）《美国国家科学院院刊》90，10769 - 10762），导致NADPH氧化和一氧化氮合成。我们研究了L-精氨酸和钙调蛋白如何控制NOS血红素铁的配体结合和电子受体特性。在没有结合钙调蛋白的情况下，通过血红素自旋平衡向高自旋状态的底物依赖性转变测定，三价铁NOS对L-精氨酸的Kd为0.6微摩尔。L-精氨酸的结合使三价铁NOS血红素对氰化物的亲和力降低了8倍。一氧化碳与无底物的二价铁NOS的结合速率为2×10⁵ M⁻¹ s⁻¹；当结合L-精氨酸时，该速率降低了12倍。相反，结合的钙调蛋白对氰化物或一氧化碳与NOS血红素的结合没有显著影响，也没有改变NOS对L-精氨酸的结合亲和力。用NADPH对结合钙调蛋白且不含L-精氨酸的NOS进行厌氧滴定导致血红素铁不完全还原；只有在添加L-精氨酸的情况下才能实现完全还原。因此，我们的数据表明L-精氨酸至少通过两种方式控制NOS血红素铁的反应性：1）它通过在血红素附近的远端口袋中结合来减缓配体相互作用，2）它似乎还增加了铁的还原电位。相比之下，结合的钙调蛋白不会改变NOS对L-精氨酸或血红素配体的亲和力，可能仅作为一个开关，使电子能够从黄素结构域传递到血红素铁上。
