Probing soluble guanylate cyclase activation by CO and YC-1 using resonance Raman spectroscopy.
Probing soluble guanylate cyclase activation by CO and YC-1 using resonance Raman spectroscopy.
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DOI：
10.1021/bi902214j
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发表时间：
2010-05-11
期刊：
影响因子：
2.9
通讯作者：
Spiro, Thomas G.
中科院分区：
文献类型：
--
作者：
Ibrahim, Mohammed;Derbyshire, Emily R.;Marletta, Michael A.;Spiro, Thomas G.
可溶性鸟苷酸环化酶（sGC）被CO弱激活，但被YC-1与sGC-CO复合物的结合显著激活。在本报告中，使用共振拉曼（RR）光谱研究选定的sGC变体。添加YC-1的sGC-CO复合物的RR频带分配给乙烯基和丙酸酯血红素取代基的强度图案的改变，这表明它们所连接的吡咯环的倾斜的变化。YC-1还将νFeC和νCO带的RR强度分别从473和1985 cm−1移动到487和1969 cm−1，并在521 cm−1处诱导出额外的νFeC带，近端血红素口袋（P118 A）或远端血红素口袋中的定点变异（V5 Y和I149 Y）降低YC-1激活的程度，沿着473 cm-1谱带强度。这些活性较低的sGC变体在493 cm−1处显示另一个νFeC带，其对YC-1添加不敏感，并且归因于不能被变构激活剂激活的蛋白质。结果与YC-1与sGC-CO结合导致激活蛋白质的构象变化的模型一致。具体而言，YC-1结合通过外周非键合接触改变血红素几何形状，并且还减轻了固有的电子效应，该电子效应减少了天然YC-1响应蛋白中的FeCO背键。这种电子效应可能涉及通过氢键接触中和血红素丙酸盐，或通过远端半胱氨酸残基负极化。YC-1结合还使Fe-组氨酸键应变，导致除了构象上不同的6-配位sGC-C 0群体之外的5-配位sGC-C 0群体。sGC变体中YC-1活化的丧失可能涉及血红素-蛋白质接触的减弱，这被认为对YC-1诱导的构象变化至关重要。
Soluble guanylate cyclase (sGC) is weakly activated by CO but is significantly activated by the binding of YC-1 to the sGC-CO complex. In this report resonance Raman (RR) spectroscopy was used to study selected sGC variants. Addition of YC-1 to the sGC-CO complex alters the intensity pattern of RR bands assigned to the vinyl and propionate heme substituents, suggesting changes in the tilting of the pyrrole rings to which they are attached. YC-1 also shifts the RR intensity of the νFeC and νCO bands from 473 and 1985 cm−1 to 487 and 1969 cm−1, respectively, and induces an additional νFeC band, at 521 cm−1, assigned to 5-coordinate heme-CO. Site-directed variants in the proximal heme pocket (P118A) or in the distal heme pocket (V5Y and I149Y) reduce the extent of YC-1 activation, along with the 473 cm−1 band intensity. These lower activity sGC variants display another νFeC band at 493 cm−1 which is insensitive to YC-1 addition and is attributed to protein that cannot be activated by the allosteric activator. The results are consistent with a model in which YC-1 binding to sGC-CO results in a conformational change that activates the protein. Specifically, YC-1 binding alters the heme geometry via peripheral non-bonded contacts, and also relieves an intrinsic electronic effect that diminishes FeCO backbonding in the native, YC-1 responsive protein. This electronic effect might involve neutralization of the heme propionates via H-bond contacts, or negative polarization by a distal cysteine residue. YC-1 binding also strains the Fe-histidine bond, leading to a population of 5-coordinate sGC-CO in addition to a conformationally distinct population of 6-coordinate sGC-CO. The loss of YC-1 activation in the sGC variants might involve a weakening of the heme-protein contacts which are thought to be critical to a YC-1-induced conformational change.
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