七种天然二氧化碳固定途径之一,厌氧Wood-Ljungdahl途径(WLP)在产生CO作为代谢中间体方面是独特的,通过有机金属中间体操作,以及在保存(与利用)净ATP方面。WLP中的关键酶是乙酰辅酶A合酶(ACS),使用活性位点[2Ni-4Fe-4S]簇(A簇),CO隧道,和有机金属(Ni-CO,镍-甲基,和Ni-乙酰基)反应序列以产生乙酰辅酶A。在这里我们揭示了一个壁龛,连接隧道和A集群,对于WLP的CO2固定和自养生长至关重要。体外光谱学,动力学,绑定,体内生长实验表明,在壁龛的一个壁上的Phe229A取代使CO亲和力降低了30倍,并消除了自养生长;但是,aF229W取代增强CO结合80倍。我们的结果表明,壁龛的结构经过精细调整,可以在A簇附近浓缩CO;在催化过程中保护ACS免受CO损失,为抑制性CO提供避风港,并稳定CO结合的Nip位点的四面体配位。导演,集中,壁龛的保护作用解释了F209A无法自养生长。壁龛还可以帮助解释当前有关ACS是否通过随机或有序机制结合CO和甲基的争议。我们的工作重新定义了我们历史上所说的金属中心“活性位点”。壁龛对于酶功能至关重要,因此我们认为它是活性位点的一部分。社区现在应该在所有“气体处理”金属酶中寻找这样的凹室。
One of the seven natural CO2 fixation pathways, the anaerobic Wood-Ljungdahl pathway (WLP) is unique in generating CO as a metabolic intermediate, operating through organometallic intermediates, and in conserving (versus utilizing) net ATP. The key enzyme in the WLP is acetyl-CoA synthase (ACS), which uses an active site [2Ni-4Fe-4S] cluster (A-cluster), a CO tunnel, and an organometallic (Ni-CO, Ni-methyl, and Ni-acetyl) reaction sequence to generate acetyl-CoA. Here, we reveal that an alcove, which interfaces the tunnel and the A-cluster, is essential for CO2 fixation and autotrophic growth by the WLP. In vitro spectroscopy, kinetics, binding, and in vivo growth experiments reveal that a Phe229A substitution at one wall of the alcove decreases CO affinity thirty-fold and abolishes autotrophic growth; however, a F229W substitution enhances CO binding 80-fold. Our results indicate that the structure of the alcove is exquisitely tuned to concentrate CO near the A-cluster; protect ACS from CO loss during catalysis, provide a haven for inhibitory CO, and stabilize the tetrahedral coordination at the Nip site where CO binds. The directing, concentrating, and protective effects of the alcove explain the inability of F209A to grow autotrophically. The alcove also could help explain current controversies over whether ACS binds CO and methyl through a random or ordered mechanism. Our work redefines what we historically refer to as the metallocenter \"active site\". The alcove is so crucial for enzymatic function that we propose it is part of the active site. The community should now look for such alcoves in all \"gas handling\" metalloenzymes.