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首页> 外文期刊>Biochemistry >Differentiation of acetylene-reduction sites by stereoselective proton addition during Azotobacter vinelandii nitrogenase-catalyzed C2D2 reduction.
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Differentiation of acetylene-reduction sites by stereoselective proton addition during Azotobacter vinelandii nitrogenase-catalyzed C2D2 reduction.

机译：在固氮菌固氮酶催化的C2D2还原过程中，通过添加立体选择性质子来区分乙炔还原位点。

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The interactions of acetylene with its binding site(s) on the FeMo cofactor of the MoFe protein of Azotobacter vinelandii nitrogenase were probed using C(2)D(2). Specifically, the effects of changing the C(2)D(2) concentration, electron flux, pH, or the individual presence of N(2), ethylene, or CO on the formation of both cis- and trans-1,2-ethylene-d(2) from C(2)D(2) were measured. A hypothesis, involving two acetylene-reduction sites, was developed to explain the changes observed in the stereoselective protonation during both substrate-concentration-dependent and electron-flux-dependent C(2)D(2) reduction. One of these sites is a higher-affinity acetylene-binding site that produces only cis-1,2-ethylene-d(2) from C(2)D(2). The other is a lower-affinity acetylene-binding site, which produces both cis- and trans-1,2-ethylene-d(2). Added N(2) specifically inhibited the production of cis-1,2-ethylene-d(2) from C(2)D(2), which indicates that N(2) binds to (and is reduced at) the higher-affinity acetylene-binding site. High concentrations of added ethylene behaved like very high concentrations of acetylene and inhibited both the electron flux flowing through the enzyme and cis-isomer formation. Added CO, at very low concentrations, did not affect the relative distribution of cis- and trans-isomers, indicating a separate CO-binding site. The results of pH-dependence experiments showed that substrate inhibition at high C(2)D(2) concentrations is enhanced under acidic conditions but is absent under basic conditions and suggest that a low proton flux has a similar impact to that of a low electron flux; both inhibit cis-1,2-ethylene-d(2) formation selectively. Apparently, the factors affecting stereoselective protonation during C(2)D(2) reduction could be the same as those that perturb protonation of the FeMo cofactor when acetylene is reduced. The observed nitrogenase-catalyzed production of ethylene-d(1) from C(2)D(2) implicates a reversible protonation step in the mechanistic pathway.
机译：使用C（2）D（2）探测了乙炔与其在葡萄固氮固氮酶MoFe蛋白的FeMo辅因子上的结合位点之间的相互作用。具体来说，改变C（2）D（2）的浓度，电子通量，pH或N（2），乙烯或CO的单独存在对顺式和反式1,2-形成的影响测量了C（2）D（2）的乙烯-d（2）。一个假说，涉及两个乙炔还原站点，被开发来解释观察到的立体选择性质子化过程中底物浓度依赖性和电子通量依赖性C（2）D（2）还原过程中的变化。这些位点之一是更高亲和力的乙炔结合位点，它仅从C（2）D（2）产生顺式1，2-乙烯-d（2）。另一个是较低亲和力的乙炔结合位点，可同时产生顺式和反式1,2-乙烯-d（2）。添加N（2）会特异性抑制C（2）D（2）产生顺式1，2-乙烯-d（2），这表明N（2）与较高的C结合（并在其上还原）。亲和乙炔结合位点。高浓度添加的乙烯表现出与极高浓度的乙炔一样的行为，并抑制了流过酶的电子通量和顺式异构体的形成。以非常低的浓度添加的CO不会影响顺式和反式异构体的相对分布，表明存在单独的CO结合位点。 pH依赖性实验的结果表明，在酸性条件下，在高C（2）D（2）浓度下底物抑制作用增强，而在碱性条件下则不存在，这表明低质子通量与低电子通量具有相似的影响通量两者均选择性抑制顺式1，2-乙烯-d（2）的形成。显然，在C（2）D（2）还原过程中影响立体选择性质子化的因素可能与在乙炔还原时扰动FeMo辅因子的质子化的因素相同。从C（2）D（2）观察到的固氮酶催化的乙烯-d（1）的生产在机械途径中涉及可逆的质子化步骤。

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