EPR and Electron Nuclear Double Resonance spectroscopies here characterize CO binding to the active-site A cluster of wild-type (WT) Acetyl-CoA Synthase (ACS) and two variants, F229W and F229A. The A-cluster binds CO to a proximal Ni (Nip) that bridges a [4Fe-4S] cluster and a distal Nid. An alcove seen in the ACS crystal structure near the A-cluster, defined by hydrophobic residues including F229, forms a cage surrounding a Xe mimic of CO. Previously, we only knew WT ACS bound a single CO to form the Ared-CO intermediate, containing Nip(I)-CO with CO located on the axis of the dz2 odd-electron orbital (g>g||~2). Here, the two-dimensional field-frequency pattern of 2K-35 GHz 13C-ENDOR spectra collected across the Ared-CO EPR envelope reveals a second CO bound in the dz2 orbital's equatorial plane. This WT A-cluster conformer dominates the nearly-conservative F229W variant, but 13C-ENDOR reveals a minority "A" conformation with (g||>g~2) characteristic of a 'cloverleaf' (eg. dx2-y2) odd-electron orbital, with Nip binding two, apparently 'in-plane' CO. Disruption of the alcove through introduction of the smaller alanine residue in the F229A variant diminishes conversion to Ni(I) ~tenfold and introduces extensive cluster flexibility. 13C-ENDOR shows the F229A cluster is mostly (60%) in the "A" conformation, but with ~20% each of the WT conformer and an "O" state in which dz2 Nip(I) (g>g||~2) surprisingly lacks CO. This paper thus demonstrates the importance of an intact alcove in forming and stabilizing the Ni(I)-CO intermediate in the Wood-Ljungdahl pathway of anaerobic CO and CO2 fixation.

中文翻译：

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13C 电子核双共振光谱显示乙酰辅酶 A 合酶以多种结合模式结合两种底物 CO 并揭示 CO 结合“壁龛”的重要性

EPR 和电子核双共振光谱在此表征 CO 与活性位点 A 簇的野生型 (WT) 乙酰辅酶 A 合酶 (ACS) 和两个变体 F229W 和 F229A 的结合。A 簇将 CO 与近端 Ni (Nip) 结合，Ni (Nip) 桥接 [4Fe-4S] 簇和远端 Nid。在 A 簇附近的 ACS 晶体结构中看到的一个凹室，由包括 F229 在内的疏水残基定义，形成一个围绕着 CO 的 Xe 模拟物的笼。以前，我们只知道 WT ACS 结合单个 CO 以形成 Ared-CO 中间体，包含 Nip(I)-CO，CO 位于 dz2 奇电子轨道的轴上 (g>g||~2)。在这里，通过 Ared-CO EPR 包络收集的 2K-35 GHz 13C-ENDOR 光谱的二维场频模式揭示了 dz2 轨道赤道平面中的第二个 CO 边界。这种 WT A 簇构象支配着近乎保守的 F229W 变体，但 13C-ENDOR 揭示了少数“A”构象，具有 (g||>g~2) 'cloverleaf'（例如 dx2-y2）奇数的特征-电子轨道，与 Nip 结合两个，显然是“平面内”CO。通过在 F229A 变体中引入较小的丙氨酸残基来破坏壁龛，减少了向 Ni(I) 的转化~十倍，并引入了广泛的簇灵活性。13C-ENDOR 显示 F229A 簇大部分 (60%) 处于“A”构象，但各有约 20% 的 WT 构象异构体和“O”状态，其中 dz2 Nip(I) (g>g|| ~2) 令人惊讶的是缺乏二氧化碳。