Duan Jifu, Rutz Andreas, Kawamoto Akihiro, Naskar Shuvankar, Edenharter Kristina, Leimkühler Silke, Hofmann Eckhard, Happe Thomas, Kurisu Genji
Photobiotechnology, Faculty of Biology and Biotechnology, Ruhr University Bochum, Bochum 44801, Germany.
Laboratory for Protein Crystallography, Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan.
Proc Natl Acad Sci U S A. 2025 Jan 21;122(3):e2416233122. doi: 10.1073/pnas.2416233122. Epub 2025 Jan 13.
[FeFe]-hydrogenases catalyze the reversible two-electron reduction of two protons to molecular hydrogen. Although these enzymes are among the most efficient H-converting biocatalysts in nature, their catalytic cofactor (termed H-cluster) is irreversibly destroyed upon contact with dioxygen. The [FeFe]-hydrogenase CbA5H from has a unique mechanism to protect the H-cluster from oxygen-induced degradation. The protective strategy of CbA5H was proposed based on a partial protein structure of CbA5H's oxygen-shielded form. Here, we present a cryo-EM structure of 2.2 Å resolution from the entire enzyme in its dimeric and active state and elucidate the structural parameters of the reversible cofactor protection mechanism. We found that both subunits of the homodimeric structure of CbA5H have a Zn-binding four-helix domain, which does not play a role in electron transport as described for other complex protein structures. Biochemical data instead confirm that two [4Fe-4S] clusters are responsible for electron transfer in CbA5H, while the identified zinc atom is critical for oligomerization and protein stability.
[铁铁]氢化酶催化两个质子可逆地双电子还原为分子氢。尽管这些酶是自然界中最有效的氢转化生物催化剂之一，但它们的催化辅因子（称为H簇）在与 dioxygen 接触时会不可逆地被破坏。来自 的[铁铁]氢化酶CbA5H具有独特的机制来保护H簇免受氧诱导的降解。CbA5H的保护策略是基于CbA5H氧屏蔽形式的部分蛋白质结构提出的。在这里，我们展示了处于二聚体和活性状态的整个酶的2.2 Å分辨率冷冻电镜结构，并阐明了可逆辅因子保护机制的结构参数。我们发现CbA5H同二聚体结构的两个亚基都有一个锌结合四螺旋结构域，该结构域在电子传递中不像其他复杂蛋白质结构那样起作用。相反，生化数据证实两个[4Fe-4S]簇负责CbA5H中的电子转移，而鉴定出的锌原子对寡聚化和蛋白质稳定性至关重要。
