Max-Planck-Institut für Bioanorganische Chemie, Stiftstrasse 34-36, Mülheim an der Ruhr, Germany.
Biochemistry. 2010 Oct 19;49(41):8873-81. doi: 10.1021/bi1006546.
The [NiFe] hydrogenase (Hase I) involved in the aerobic respiration of the hyperthermophilic bacterium Aquifex aeolicus shows increased oxygen tolerance and thermostability and can form very stable films on pyrolytic graphite electrodes. Oxygen-tolerant enzymes, like the ones from A. aeolicus and Ralstonia eutropha, are reported to be insensitive to CO inhibition. This is in contrast to known and well-characterized (oxygen-sensitive) hydrogenases, for which carbon monoxide is a competitive inhibitor. In this study, the interaction of Hase I from A. aeolicus with CO is examined using in situ infrared electrochemistry and time-resolved FTIR spectroscopy. We could observe the formation of a CO adduct state, a finding that set the grounds to investigate the affinity of an O(2)-tolerant enzyme for binding CO as well as the reversibility of this process. In the case of A. aeolicus, this extrinsic CO is shown to be weakly attached and the adduct state is light-sensitive at low temperatures. The energetic parameters for the rebinding of CO at the active site were estimated from the rate constants of this process after photolysis and the results compared to those obtained for standard hydrogenases. Formation of a weak Ni-CO bond in the active site of Hase I most likely results from the different interaction of this enzyme with inhibitors and/or different active site electronic properties to which non standard amino acid residues in the vicinity of the active site might contribute.
参与嗜热菌 Aquifex aeolicus 需氧呼吸的 [NiFe]氢化酶（Hase I）具有更高的耐氧性和热稳定性，并且可以在热解石墨电极上形成非常稳定的膜。据报道，耐氧酶，如来自 A. aeolicus 和 Ralstonia eutropha 的酶，对 CO 抑制不敏感。这与已知的和特征良好的（氧敏感）氢化酶形成对比，对于后者，一氧化碳是竞争性抑制剂。在这项研究中，使用原位红外电化学和时间分辨傅里叶变换红外光谱研究了来自 A. aeolicus 的 Hase I 与 CO 的相互作用。我们可以观察到 CO 加合物状态的形成，这一发现为研究 O(2)-耐受酶结合 CO 的亲和力以及该过程的可逆性奠定了基础。在 A. aeolicus 的情况下，证明这种外来的 CO 是弱结合的，并且在低温下，加合物状态对光敏感。通过光解后该过程的速率常数来估计 CO 在活性位点重新结合的能量参数，并将结果与标准氢化酶的结果进行比较。在 Hase I 的活性位点形成弱 Ni-CO 键很可能是由于该酶与抑制剂的不同相互作用和/或活性位点电子性质的不同，这可能与活性位点附近的非标准氨基酸残基有关。
