13C NMR characterization of an exchange reaction between CO and CO2 catalyzed by carbon monoxide dehydrogenase.
13C NMR characterization of an exchange reaction between CO and CO2 catalyzed by carbon monoxide dehydrogenase.
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DOI：
10.1021/bi8004522
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发表时间：
2008-07-01
期刊：
影响因子：
2.9
通讯作者：
Ragsdale, Stephen W.
中科院分区：
文献类型：
--
作者：
Seravalli, Javier;Ragsdale, Stephen W.
一氧化碳脱氢酶 (CODH) 在镍-铁-硫簇（C 簇）中催化 CO 可逆氧化为 CO2。 CO 氧化遵循乒乓机制，涉及 C 簇的双电子还原，然后通过内部电子转移链将电子转移到外部电子受体。我们描述了 13C NMR 研究，证明了在没有外部电子受体的情况下，CO 和 CO2 之间发生 CODH 催化的稳态交换反应。该反应的特点是 13CO NMR 共振依赖 CODH 展宽；然而，13CO共振的化学位移没有变化，表明展宽处于NMR实验的慢交换极限。 13CO 谱线展宽的速率常数（20 °C 时为 1080 s−1）约等于 CO 氧化的速率常数。结论是，观察到的交换反应发生在 13CO 和 CODH 结合的 13CO2 之间，因为 13CO 谱线展宽与 pH 无关（与稳态 CO 氧化不同），因为它需要功能性 C 簇（而不是功能性 B 簇），而且 13CO2 谱线宽度不会展宽。此外，溶液中 12CO 和 13CO2 之间的稳态同位素交换反应以与 CO2 还原相同的速率发生，这比 13CO 交换展宽的速率慢约 750 倍。 CODH 和抑制剂氰化物 (CN−) 之间的相互作用也通过 13C NMR 进行了探测。 13CN− 展宽不需要功能性 C 簇（与 13CO 不同），并且其交换速率常数比 13CO 快 30 倍。综合结果表明 13CO 交换包括 CO 迁移到 C 簇，以及 CO 氧化成 CO2，但不将 CO2 或质子释放到溶剂中。它们还提供了 CO2 结合位点和 CODH 内部质子转移网络的有力证据。 13CN−交换似乎仅监测CN−在溶液之间的移动及其与CODH的结合和释放。
Carbon monoxide dehydrogenase (CODH) catalyzes the reversible oxidation of CO to CO2 at a nickel−iron−sulfur cluster (the C-cluster). CO oxidation follows a ping-pong mechanism involving two-electron reduction of the C-cluster followed by electron transfer through an internal electron transfer chain to external electron acceptors. We describe 13C NMR studies demonstrating a CODH-catalyzed steady-state exchange reaction between CO and CO2 in the absence of external electron acceptors. This reaction is characterized by a CODH-dependent broadening of the 13CO NMR resonance; however, the chemical shift of the 13CO resonance is unchanged, indicating that the broadening is in the slow exchange limit of the NMR experiment. The 13CO line broadening occurs with a rate constant (1080 s−1 at 20 °C) that is approximately equal to that of CO oxidation. It is concluded that the observed exchange reaction is between 13CO and CODH-bound 13CO2 because 13CO line broadening is pH-independent (unlike steady-state CO oxidation), because it requires a functional C-cluster (but not a functional B-cluster) and because the 13CO2 line width does not broaden. Furthermore, a steady-state isotopic exchange reaction between 12CO and 13CO2 in solution was shown to occur at the same rate as that of CO2 reduction, which is approximately 750-fold slower than the rate of 13CO exchange broadening. The interaction between CODH and the inhibitor cyanide (CN−) was also probed by 13C NMR. A functional C-cluster is not required for 13CN− broadening (unlike for 13CO), and its exchange rate constant is 30-fold faster than that for 13CO. The combined results indicate that the 13CO exchange includes migration of CO to the C-cluster, and CO oxidation to CO2, but not release of CO2 or protons into the solvent. They also provide strong evidence of a CO2 binding site and of an internal proton transfer network in CODH. 13CN− exchange appears to monitor only movement of CN− between solution and its binding to and release from CODH.
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