INHIBITION OF NITRIC-OXIDE SYNTHASE ACTIVITY BY ZN2+ ION
INHIBITION OF NITRIC-OXIDE SYNTHASE ACTIVITY BY ZN2+ ION
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DOI：
10.1021/bi00046a015
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发表时间：
1995-11-21
期刊：
影响因子：
2.9
通讯作者：
MASTERS, BSS
中科院分区：
文献类型：
--
作者：
PERSECHINI, A;MCMILLAN, K;MASTERS, BSS
我们发现Zn ~（2+）能完全可逆地抑制神经型一氧化氮合酶（nNOS）的活性，其表观K ~（-1）为30 μ M。Zn 2+阻断nNOS中血红素铁的NADPH依赖性还原，也阻断nNOS所表现出的钙调素依赖性超氧化物介导的细胞色素c还原酶活性。然而，Zn ~（2+）离子对nNOS非钙调素依赖性直接还原细胞色素c没有明显影响。Zn ~（2+）离子在nNOS中引起的微扰差光谱的特征是在近似430 nm处出现一个峰，在近似395 nm处出现一个谷，其表观光谱结合常数为50 μ M。这些光谱的变化是一致的Zn 2+依赖的变化，在nNOS的血红素铁的自旋态平衡。L-精氨酸与nNOS结合的光谱结合常数（类似于1.5 μ M）不受50 μ M Zn 2+存在的显著影响，表明Zn 2+依赖性抑制nNOS活性不是由于干扰底物结合。nNOS吸光度的估计最大变化，在类似于418 nm的L-精氨酸依赖的转换所造成的铁血红素铁从六配位低自旋五配位高自旋增加了50%，在50 μ M的Zn 2+的存在下，这反映了增加的初始量的低自旋铁血红素铁存在。这些数据表明，锌2+依赖性抑制nNOS活性是由于结合的锌2+血红素蛋白结构域的酶和抑制是与扰动的血红素铁的环境中，似乎阻止其介导氧还原的能力。
We have found neural nitric oxide synthase (nNOS) activity to be completely and reversibly inhibited by Zn2+ ion with an apparent K-i of 30 mu M. Zn2+ blocks NADPH-dependent reduction of heme iron in nNOS and also blocks the calmodulin-dependent superoxide-mediated cytochrome c reductase activity exhibited by nNOS. However, Zn2+ ion has no apparent effect on the calmodulin-independent direct reduction of cytochrome c by nNOS. Zn2+ ion induces perturbation difference spectra in nNOS characterized by the appearance of a peak at similar to 430 nm and a trough at similar to 395 nm, with an apparent spectral binding constant of 50 mu M. These spectral changes are consistent with a Zn2+-dependent change in the spin-state equilibrium of the heme iron in nNOS. The Spectral binding constant for L-arginine binding to nNOS (similar to 1.5 mu M) is not significantly affected by the presence of 50 mu M Zn2+, indicating that Zn2+-dependent inhibition of nNOS activity is not due to interference with substrate binding. The estimated maximal change in nNOS absorbance at similar to 418 nm caused by the L-arginine-dependent conversion of the ferric heme iron from hexacoordinate low-spin to pentacoordinate high-spin is increased by 50% in the presence of 50 mu M Zn2+, which reflects the increased initial amount of low-spin ferric heme iron present. These data indicate that Zn2+-dependent inhibition of nNOS activity is due to binding of Zn2+ to the hemoprotein domain in the enzyme and that inhibition is associated with perturbations in the environment of the heme iron that appear to block its ability to mediate oxygen reduction.