CNRS, Laboratoire de Bioénergétique et Ingénierie des Protéines, Institut de Microbiologie de la Méditerranée, 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
J Am Chem Soc. 2011 Feb 2;133(4):986-97. doi: 10.1021/ja108787s. Epub 2010 Dec 22.
Hydrogenases are efficient biological catalysts of H(2) oxidation and production. Most of them are inhibited by O(2), and a prerequisite for their use in biotechnological applications under air is to improve their oxygen tolerance. We have previously shown that exchanging the residue at position 74 in the large subunit of the oxygen-sensitive [NiFe] hydrogenase from Desulfovibrio fructosovorans could impact the reaction of the enzyme with O(2) (Dementin, S.; J. Am. Chem. Soc. 2009, 131, 10156-10164; Liebgott, P. P.; Nat. Chem. Biol. 2010, 6, 63-70). This residue, a valine in the wild-type enzyme, located at the bottleneck of the gas channel near the active site, has here been exchanged with a cysteine. A thorough characterization using a combination of kinetic, spectroscopic (EPR, FTIR), and electrochemical studies demonstrates that the V74C mutant has features of the naturally occurring oxygen-tolerant membrane-bound hydrogenases (MBH). The mutant is functional during several minutes under O(2), has impaired H(2)-production activity, and has a weaker affinity for CO than the WT. Upon exposure to O(2), it is converted into the more easily reactivatable inactive form, Ni-B, and this inactive state reactivates about 20 times faster than in the WT enzyme. Control experiments carried out with the V74S and V74N mutants indicate that protonation of the position 74 residue is not the reason the mutants reactivate faster than the WT enzyme. The electrochemical behavior of the V74C mutant toward O(2) is intermediate between that of the WT enzyme from D. fructosovorans and the oxygen-tolerant MBH from Aquifex aeolicus.
氢化酶是高效的 H(2)氧化和生产生物催化剂。它们大多数都被 O(2)抑制，并且它们在空气中的生物技术应用中使用的前提是提高其耐氧性。我们之前已经表明，交换位置 74 的残基在大型亚基的氧敏感[NiFe]氢化酶从脱硫弧菌 fructosovorans 可以影响酶与 O(2)的反应（Dementin，S.；J. Am. Chem. Soc. 2009, 131, 10156-10164; Liebgott，P. P.；Nat. Chem. Biol. 2010, 6, 63-70）。这个残基，野生型酶中的缬氨酸，位于靠近活性位点的气体通道瓶颈处，已经被替换为半胱氨酸。使用动力学、光谱（EPR、FTIR）和电化学研究的组合进行了彻底的表征，证明 V74C 突变体具有天然存在的耐氧膜结合氢化酶（MBH）的特征。该突变体在 O(2)下能维持几分钟的功能，其 H(2)-生产活性受损，并且对 CO 的亲和力比 WT 弱。暴露于 O(2)时，它转化为更容易重新激活的非活性形式 Ni-B，并且这种非活性状态的重新激活比 WT 酶快约 20 倍。用 V74S 和 V74N 突变体进行的对照实验表明，位置 74 残基的质子化不是突变体比 WT 酶更快重新激活的原因。V74C 突变体对 O(2)的电化学行为介于 D. fructosovorans 的 WT 酶和 Aquifex aeolicus 的耐氧 MBH 之间。
