Zoleo Alfonso, Lippe Giovanna, Contessi Stefania, Brustolon Marina, Dabbeni-Sala Federica, Maniero Anna Lisa
Department of Chemistry, University of Padova, via Marzolo 1, Italy.
Biochemistry. 2007 Nov 20;46(46):13443-50. doi: 10.1021/bi700949e. Epub 2007 Oct 26.
The catalytic sites of beef heart mitochondrial F1-ATPase were studied by electron spin echo envelope modulation (ESEEM) spectroscopy, using Mn(II) as a paramagnetic probe, which replaces the naturally occurring Mg(II), maintaining the enzyme catalytic activity. F1-ATPase was purified from beef heart mitochondria. A protein still containing three endogenous nucleotides, named MF1(1,2), is obtained under milder conditions, whereas a harsher treatment gives a fully depleted F1, named MF1(0,0). Several samples were prepared, loading MF1(0,0) or MF1(1,2) with Mn(II) or MnIIADP in both substoichiometric and excess amounts. When MF1(1,2) is loaded with Mn(II) in a 1:0.8 ratio, the FT-ESEEM spectrum shows evidence of a nitrogen interacting with the metal, while this interaction is not present in MF1(0,0) + Mn(II) in a 1:0.8 ratio. However, when MF1(0,0) is loaded with 2.4 Mn(II), the FT-ESEEM spectrum shows a metal-nitrogen interaction resembling that present in MF1(1,2) + Mn(II) in a 1:0.8 ratio. These results strongly support the role of the metal alone in shaping and structuring the catalytic sites of the enzyme. When substoichiometric ADP is added to MF1(1,2) preloaded with 0.8 equiv of Mn(II), the ESEEM spectra show evidence of a phosphorus nucleus coupled to the metal, indicating that the nucleotide phosphate binding to Mn(II) occurs in a catalytic site. Generally, 14N coordination to the metal is clearly identified in the ESEEM spectra of all the samples containing more than one metal equivalent. One point of note is that the relevant nitrogen-containing ligand(s), responsible for the signals in the ESEEM spectra, has not yet been identified in the available X-ray structures.
利用电子自旋回波包络调制(ESEEM)光谱法,以锰(II)作为顺磁探针,对牛心线粒体F1 - ATP酶的催化位点进行了研究。锰(II)取代了天然存在的镁(II),并维持了该酶的催化活性。F1 - ATP酶从牛心线粒体中纯化得到。在较温和的条件下可获得一种仍含有三个内源性核苷酸的蛋白质,命名为MF1(1,2),而更严苛的处理则得到完全耗尽核苷酸的F1,命名为MF1(0,0)。制备了多个样品,以亚化学计量和过量的量用锰(II)或锰 - ADP加载MF1(0,0)或MF1(1,2)。当MF1(1,2)以1:0.8的比例加载锰(II)时,傅里叶变换 - ESEEM光谱显示有一个氮与金属相互作用的证据,而在以1:0.8比例的MF1(0,0) + 锰(II)中不存在这种相互作用。然而,当MF1(0,0)加载2.4个锰(II)时,傅里叶变换 - ESEEM光谱显示出一种类似于以1:0.8比例的MF1(1,2) + 锰(II)中存在的金属 - 氮相互作用。这些结果有力地支持了金属单独在塑造和构建该酶催化位点中的作用。当向预先加载了0.8当量锰(II)的MF1(1,2)中加入亚化学计量的ADP时,ESEEM光谱显示有一个磷核与金属耦合的证据,表明核苷酸磷酸与锰(II)的结合发生在催化位点。一般来说,在所有含有超过一个金属当量的样品的ESEEM光谱中都能清楚地识别出与金属的14N配位。需要注意的一点是,在现有的X射线结构中尚未确定导致ESEEM光谱中信号的相关含氮配体。