Alkyne substrate interaction within the nitrogenase MoFe protein
Alkyne substrate interaction within the nitrogenase MoFe protein
复制标题
DOI：
10.1016/j.jinorgbio.2007.05.007
复制
发表时间：
2007-11-01
影响因子：
3.9
通讯作者：
Dean, Dennis R.
中科院分区：
文献类型：
--
作者：
Dos Santos, Patricia C.;Mayer, Suzanne M.;Dean, Dennis R.
固氮酶催化N-2到氨的生物还原（固氮），以及非生理炔底物乙炔（HC=CH）的双电子还原。一个复杂的金属有机物种称为FeMo-辅因子提供的网站内的MoFe蛋白质的底物还原，但究竟在哪里以及如何基板与FeMo-辅因子相互作用仍然未知。最近的结果表明，MoFe蛋白α-70（瓦尔）残基，其侧链接近FeMo-辅因子的一个Fe-S面，在限定底物进入活性位点方面起着重要作用。例如，α-70（瓦尔）被丙氨酸取代导致较大的炔丙炔（HC= C-CH 3）的还原能力增加，而在该位置被异亮氨酸取代几乎消除了乙炔的还原能力。这些和互补的光谱研究使我们提出，短链炔的结合发生与侧结合到铁原子6内的铁钼辅因子。在本工作中，α-70（瓦尔）残基被甘氨酸取代，并且该MoFe蛋白变体显示出增加的还原末端炔、1-丁炔（HC= C-CH 2-CH 3）的能力。该蛋白质未显示出可检测到的内部炔2-丁炔（H3C-C = C-CH 3）的还原。相反，用丙氨酸取代附近的α-191（Gln）残基，结合α-70（Ala）取代，确实导致2-丁炔的显著减少，唯一的产物是2-顺式-丁烯。这些结果表明，通过固氮酶的炔的还原涉及侧结合的炔铁6内的铁钼辅因子，并不需要一个终端的酸性质子的减少。成功设计的氨基酸取代，允许有针对性的住宿的炔，否则不是固氮酶底物提供的证据，以支持当前模型的固氮酶MoFe蛋白质内的炔相互作用。(C)2007年爱思唯尔公司All rights reserved.
Nitrogenase catalyzes the biological reduction of N-2 to ammonia (nitrogen fixation), as well as the two-electron reduction of the non-physiological alkyne substrate acetylene (HC=CH). A complex metallo-organic species called FeMo-cofactor provides the site of substrate reduction within the MoFe protein, but exactly where and how substrates interact with FeMo-cofactor remains unknown. Recent results have shown that the MoFe protein alpha-70(Val) residue, whose side chain approaches one Fe-S face of FeMo-cofactor, plays a significant role in defining substrate access to the active site. For example, substitution of alpha-70(Val) by alanine results in an increased capacity for the reduction of the larger alkyne propyne (HC=C - CH3) whereas, substitution by isoleucine at this position nearly eliminates the capacity for the reduction of acetylene. These and complementary spectroscopic studies led us to propose that binding of short chain alkynes occurs with side-on binding to Fe atom 6 within FeMo-cofactor. In the present work, the alpha-70(Val) residue was substituted by glycine and this MoFe protein variant shows an increased capacity for reduction of the terminal alkyne, 1-butyne (HC=C - CH2 - CH3). This protein shows no detectable reduction of the internal alkyne 2-butyne (H3C - C=C - CH3). In contrast, substitution of the nearby alpha- 191(Gln) residue by alanine, in combination with the alpha-70(Ala) substitution, does result in significant reduction of 2-butyne, with the exclusive product being 2-cis-butene. These results indicate that the reduction of alkynes by nitrogenases involves side-on binding of the alkyne to Fe6 within FeMo-cofactor, and that a terminal acidic proton is not required for reduction. The successful design of amino acid substitutions that permit the targeted accommodation of an alkyne that otherwise is not a nitrogenase substrate provides evidence to support the current model for alkyne interaction within the nitrogenase MoFe protein. (C) 2007 Elsevier Inc. All rights reserved.