Tetrahydrobiopterin-deficient nitric oxide synthase has a modified heme environment and forms a cytochrome P-420 analogue.
Tetrahydrobiopterin-deficient nitric oxide synthase has a modified heme environment and forms a cytochrome P-420 analogue.
复制标题
DOI：
10.1021/bi00021a020
复制
发表时间：
1995-05
期刊：
影响因子：
2.9
通讯作者：
Jianling Wang;D. Stuehr;Denis L. Rousseau
中科院分区：
文献类型：
--
作者：
Jianling Wang;D. Stuehr;Denis L. Rousseau
光学吸收和共振拉曼光谱的神经元型一氧化氮合酶（b-NOS）的情况下分离的四氢生物蝶呤证明，酶制剂是非常不稳定的。这种不稳定形式的酶具有类似于细胞色素P-420 cam的性质，细胞色素P-420 cam是细胞色素P-450 cam的非活性形式。虽然半胱氨酸被保存为铁和亚铁形式的不稳定的b-NOS的近端配体，缺乏四氢生物蝶呤显着增加六配位低自旋部分的血红素含量，导致酶活性的损失。在添加CO时，不稳定的b-NOS从在443 nm处显示Soret吸收最大值的物质转化为在421 nm处具有Soret最大值的物质，如对于稳定的b-NOS和细胞色素P-450 cam的CO加合物所报道的。421-nm形式的共振拉曼光谱与低pH下CO结合的肌红蛋白和CO结合的细胞色素P-420 cam的共振拉曼光谱相同。血红素在这种形式的酶是由一个较弱的配体比硫醇盐协调;组氨酸协调的CO结合形式的P-420样物种的NOS是与所有可用的数据一致。还检测到类似的不稳定形式的巨噬细胞（i-NOS）酶。不仅NOS的血红素口袋具有与稳定形式的细胞色素P-450相同的配位，而且部分变性形式具有与细胞色素P-420（细胞色素P-450的无活性形式）相同的性质。在其他可能的作用中，四氢生物蝶呤可能在活性酶的稳定中发挥重要作用。
Optical absorption and resonance Raman spectra of neuronal nitric oxide synthase (b-NOS) isolated in the absence of tetrahydrobiopterin demonstrate that the enzyme preparation is very unstable. This unstable form of the enzyme has properties analogous to those of cytochrome P-420cam, an inactive form of cytochrome P-450cam. Although cysteine is preserved as the proximal ligand in both the ferric and ferrous forms of unstable b-NOS, the lack of tetrahydrobiopterin significantly increases the hexacoordinate low-spin fraction of the heme content, resulting in a loss of the enzymatic activity. Upon the addition of CO, the unstable b-NOS converts from a species exhibiting a Soret absorption maximum at 443 nm, as reported for the CO adducts of stable b-NOS and cytochrome P-450cam, to a species with a Soret maximum at 421 nm. The resonance Raman spectrum of the 421-nm form is the same as those of CO-bound myoglobin at low pH and CO-bound cytochrome P-420cam. The heme in this form of the enzyme is coordinated by a weaker ligand than thiolate; histidine coordination in the CO-bound form of the P-420-like species of NOS is consistent with all of the available data. A similar unstable form of the macrophage (i-NOS) enzyme was also detected. Not only does the heme pocket of NOS have the same coordination as cytochrome P-450 in its stable form, but the partially denatured form has the same properties as cytochrome P-420, the inactive form of cytochrome P-450. Among other possible roles, tetrahydrobiopterin may play a significant role in the stabilization of the active enzyme.