In vitro maturation of fully active [FeFe]- hydrogenase in a defined system including the iron carrier NfuA

• 成果类型: Article
• 署名作者: Marlott, Alexander; Pagnier, Adrien; Shepard, Eric M.; Balci, Batuhan; Teye, Abraham; Warui, Douglas M.; Yang, Hao; Drena, Alex; Booker, Squire J.; Hoffman, Brian M.; Broderick, William E.; Broderick, Joan B.
• 署名单位: Montana State University System; Montana State University Bozeman; Pennsylvania Commonwealth System of Higher Education (PCSHE); Pennsylvania State University; Pennsylvania State University - University Park; Howard Hughes Medical Institute; Pennsylvania Commonwealth System of Higher Education (PCSHE); Pennsylvania State University; Pennsylvania State University - University Park; Northwestern University
• 刊物名称: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• ISSN/ISSBN: 0027-8911
• DOI: 10.1073/pnas.2517347122
• 发表日期: 2025-09-30
• 关键词: radical sam; h-cluster; escherichia-coli; carbon-monoxide; sulfur cluster; catalytic-properties; 4fe-4s cluster; protein hydf; enzyme; COORDINATION

摘要

The [FeFe]- hydrogenase employs an active-site 6Fe H-cluster to catalyze the reversible reduction of protons to H2. A [4Fe-4S] subcluster of the H-cluster is synthesized by housekeeping iron-sulfur cluster assembly machinery, and then dedicated hydrogenase maturation enzymes, together with components of the glycine cleavage system, build and deliver a [2Fe] subcluster to generate the full H-cluster. Here, we report that the Escherichia coli iron-sulfur carrier protein NfuA supports in vitro maturation of fully active [FeFe]- hydrogenase, with H2 production rates comparable to that of the in vivo-matured Chlamydomonas reinhardtii [FeFe]- hydrogenase (CrHydA). Inclusion of NfuA in the in vitro maturation process improves its efficacy by delivering the iron essential for formation of the [FeII(cys)(CN)(CO)2]- synthon at the dangler iron site of the HydG auxiliary cluster. NfuA serves an additional role in reconstituting and maintaining the catalytically essential iron-sulfur clusters on the maturase enzymes HydE, HydF, and HydG. Further inclusion of a high CO affinity myoglobin variant (MbH64L) sequesters free CO generated during the maturation process, minimizing formation of the CO-inhibited Hox-CO enzyme state, significantly increasing hydrogenase activity. The addition of NfuA and MbH64L to the fully defined maturation system thus results in an in vitro [FeFe]- hydrogenase maturation system that generates highly active enzyme while providing insights into factors important to in vivo maturation.