Funk Tobias, Gu Weiwei, Friedrich Stephan, Wang Hongxin, Gencic Simonida, Grahame David A, Cramer Stephen P
Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA.
J Am Chem Soc. 2004 Jan 14;126(1):88-95. doi: 10.1021/ja0366033.
The 5-subunit-containing acetyl-CoA decarbonylase/synthase (ACDS) complex plays an important role in methanogenic Archaea that convert acetate to methane, by catalyzing the central reaction of acetate C-C bond cleavage in which acetyl-CoA serves as the acetyl donor substrate reacting at the ACDS beta subunit active site. The properties of Ni in the active site A-cluster in the ACDS beta subunit from Methanosarcina thermophila were investigated. A recombinant, C-terminally truncated form of the beta subunit was employed, which mimics the native subunit previously isolated from the ACDS complex, and contains an A-cluster composed of an [Fe(4)S(4)] center bridged to a binuclear Ni-Ni site. The electronic structures of these two Ni were studied using L-edge absorption and X-ray magnetic circular dichroism (XMCD) spectroscopy. The L-edge absorption data provided evidence for two distinct Ni species in the as-isolated enzyme, one with low-spin Ni(II) and the other with high-spin Ni(II). XMCD spectroscopy confirmed that the species producing the high-spin signal was paramagnetic. Upon treatment with Ti(3+) citrate, an additional Ni species emerged, which was assigned to Ni(I). By contrast, CO treatment of the reduced enzyme converted nearly all of the Ni in the sample to low-spin Ni(II). The results implicate reaction of a high-spin tetrahedral Ni site with CO to form an enzyme-CO adduct transformed to a low-spin Ni(II) state. These findings are discussed in relation to the mechanism of C-C bond activation, in connection with the model of the beta subunit A-cluster developed from companion Ni and Fe K edge, XANES, and EXAFS studies.
含5个亚基的乙酰辅酶A脱羧酶/合酶（ACDS）复合物在将乙酸盐转化为甲烷的产甲烷古菌中起着重要作用，它催化乙酸盐C-C键裂解的核心反应，其中乙酰辅酶A作为乙酰供体底物在ACDSβ亚基活性位点发生反应。研究了嗜热甲烷八叠球菌ACDSβ亚基活性位点A簇中镍的性质。使用了一种重组的、C端截短形式的β亚基，它模拟了先前从ACDS复合物中分离出的天然亚基，并包含一个由桥接到双核Ni-Ni位点的[Fe(4)S(4)]中心组成的A簇。利用L边吸收和X射线磁圆二色性（XMCD）光谱研究了这两种镍的电子结构。L边吸收数据为分离出的酶中两种不同的镍物种提供了证据，一种是低自旋Ni(II)，另一种是高自旋Ni(II)。XMCD光谱证实产生高自旋信号的物种是顺磁性的。用柠檬酸钛(3+)处理后，出现了另一种镍物种，被归为Ni(I)。相比之下，用CO处理还原后的酶几乎将样品中的所有镍转化为低自旋Ni(II)。结果表明，高自旋四面体镍位点与CO反应形成酶-CO加合物，然后转变为低自旋Ni(II)状态。结合从相关的Ni和Fe K边、XANES和EXAFS研究中建立的β亚基A簇模型，讨论了这些发现与C-C键活化机制的关系。
