Poster Presentation 21st International Conference on Biological Inorganic Chemistry 2025

Cryo-EM structure of the ATP driven Methyl coenzyme M reductase activation complex (#433)

Anuj Kumar 1 , Fidel Ramírez-Amador 1 , Sophia Paul 1 , Christian Lorent 2 , Stefan Bohn 3 , Georg Hochberg 4 , Silvan Scheller 5 , Sven T. Stripp 2 , Jan Michael Schuller 1
  1. Center for Synthetic Microbiology (SYNMIKRO), Marburg, Marburg, HESSE, Germany
  2. Division of Physical Chemistry, Technische Universität Berlin, Berlin, Germany
  3. Helmholtz Munich Cryo-Electron Microscopy Platform, Helmholtz Munich, Munich, Germany
  4. Max Planck Institute for Terrestrial Microbiology, Marburg, Germany
  5. Department of Bioproducts and Biosystems, School of Chemical Engineering, Aalto University, Espoo, Espoo, Finland

Methyl-coenzyme M reductase (MCR) is responsible for the production of nearly all biologically generated methane1. MCR represents the most abundant enzymes on Earth, playing crucial roles in the global carbon cycle and significantly influencing climate change2. The catalytic site of the MCR complex comprises of an F430 coenzyme, a porphyrin-based cofactor with a central nickel ion that is active exclusively in the Ni(I) state3,4. How methanogenic archaea perform the bioenergetically challenging reductive activation of F430 remains a major gap in understanding one of the most ancient systems in nature. In the mcrBDCGA operon of Methanococcus maripaludis, McrC was identified in a multi-protein system potentially activating the MCR5. By integrating a Twin-Strep (TS) tag at the genome level in the N-terminus of McrC, we managed to co-purify MCR along with the previously uncharacterized methanogenic marker proteins Mmp7, Mmp17, Mmp3 and the A2 component – an ATPase. We demonstrate that the complex can be activated in vitro in a strictly ATP-dependent manner, enabling the production of CH4. To resolve the molecular details of the assembly and its activation process, we used redox-controlled cryo-EM to determine the single-particle structure of the protein complex exhibiting different functional states with local resolutions reaching 1.8 to 2.1 Å. Strikingly, our structure revealed three complex iron-sulfur (FeS) clusters with a topology similar to the L-cluster [8Fe-9S-C], which is a maturation intermediate of nitrogenase catalytic cofactor6,7. Within the MCR activation complex, these FeS clusters form an unprecedented electron transfer pathway toward F430, indicating their role in relaying extremely low potential electrons to reduce the nickel ion. Overall, our structural and biochemical findings offer novel insights into the ATP-dependent activation mechanism of MCR and prospects on the early evolution of nitrogenase.

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