Koo Jamin, Shiigi Stacey, Rohovie Marcus, Mehta Kunal, Swartz James R
From the Departments of Chemical Engineering and.
Bioengineering, Stanford University, Stanford, California 94305.
J Biol Chem. 2016 Oct 7;291(41):21563-21570. doi: 10.1074/jbc.M116.737122. Epub 2016 Jul 19.
[FeFe] hydrogenases catalyze rapid H production but are highly O-sensitive. Developing O-tolerant enzymes is needed for sustainable H production technologies, but the lack of a quantitative and predictive assay for O tolerance has impeded progress. We describe a new approach to provide quantitative assessment of O sensitivity by using an assay employing ferredoxin NADP reductase (FNR) to transfer electrons from NADPH to hydrogenase via ferredoxins (Fd). Hydrogenase inactivation is measured during H production in an O-containing environment. An alternative assay uses dithionite (DTH) to provide reduced Fd. This second assay measures the remaining hydrogenase activity in periodic samples taken from the NADPH-driven reaction solutions. The second assay validates the more convenient NADPH-driven assay, which better mimics physiological conditions. During development of the NADPH-driven assay and while characterizing the Clostridium pasteurianum (Cp) [FeFe] hydrogenase, CpI, we detected significant rates of direct electron loss from reduced Fd to O However, this loss does not interfere with measurement of first order hydrogenase inactivation, providing rate constants insensitive to initial hydrogenase concentration. We show increased activity and O tolerance for a protein fusion between Cp ferredoxin (CpFd) and CpI mediated by a 15-amino acid linker but not for a longer linker. We suggest that this precise, solution phase assay for [FeFe] hydrogenase O sensitivity and the insights we provide constitute an important advance toward the discovery of the O-tolerant [FeFe] hydrogenases required for photosynthetic, biological H production.
[铁铁]氢化酶能催化快速产氢，但对氧气高度敏感。可持续产氢技术需要开发耐氧的酶，但缺乏一种用于定量和预测耐氧性的检测方法阻碍了这一进展。我们描述了一种新方法，通过使用一种检测方法来定量评估氧气敏感性，该方法利用铁氧化还原蛋白NADP还原酶（FNR）通过铁氧化还原蛋白（Fd）将电子从NADPH转移到氢化酶。在含氧量环境中产氢过程中测量氢化酶失活情况。另一种检测方法使用连二亚硫酸盐（DTH）来提供还原态的Fd。第二种检测方法测量从NADPH驱动的反应溶液中定期取样的剩余氢化酶活性。第二种检测方法验证了更方便的NADPH驱动检测方法，该方法能更好地模拟生理条件。在开发NADPH驱动检测方法以及表征巴氏梭菌（Cp）[铁铁]氢化酶CpI的过程中，我们检测到还原态的Fd向氧气直接电子损失的显著速率。然而，这种损失并不干扰一级氢化酶失活的测量，从而提供对初始氢化酶浓度不敏感的速率常数。我们发现由15个氨基酸连接子介导的Cp铁氧化还原蛋白（CpFd）和CpI之间的蛋白质融合具有更高的活性和耐氧性，但对于更长的连接子则不然。我们认为，这种针对[铁铁]氢化酶氧气敏感性的精确的溶液相检测方法以及我们提供的见解，是朝着发现光合生物产氢所需的耐氧[铁铁]氢化酶迈出的重要一步。
