On the Mechanism of Biological Methane Formation: Structural Evidence for Conformational Changes in Methyl-coenzyme M Reductase upon Substrate Binding Wolfgang Grabarse 1,2 , Felix Mahlert 2 , Evert C. Duin 2 , Marcel Goubeaud 2 , Seigo Shima 2 , Rudolf K. Thauer 2 , Victor Lamzin 3 and Ulrich Ermler 1 * 1 Max-Planck-Institut fu Èr Biophysik, Heinrich-Hoffmann- Straûe 7, 60528 Frankfurt Germany 2 Max-Planck-Institut fu Èr terrestrische Mikrobiologie Karl-von-Frisch-Straûe 35043 Marburg, Germany 3 EMBL Hamburg outstation Notkestr 85, 22603 Hamburg Germany Methyl-coenzyme M reductase (MCR) catalyzes the ®nal reaction of the energy conserving pathway of methanogenic archaea in which methyl- coenzyme M and coenzyme B are converted to methane and the hetero- disul®de CoM-S-S-CoB. It operates under strictly anaerobic conditions and contains the nickel porphinoid F 430 which is present in the nickel (I) oxidation state in the active enzyme. The known crystal structures of the inactive nickel (II) enzyme in com- plex with coenzyme M and coenzyme B (MCR-ox1-silent) and in complex with the heterodisul®de CoM-S-S-CoB (MCR-silent) were now re®ned at 1.16 A Ê and 1.8 A Ê resolution, respectively. The atomic resolution structure of MCR-ox1-silent describes the exact geometry of the cofactor F 430 , of the active site residues and of the modi®ed amino acid residues. More- over, the observation of 18 Mg 2 and 9 Na  ions at the protein surface of the 300 kDa enzyme speci®es typical constituents of binding sites for either ion. The MCR-silent and MCR-ox1-silent structures differed in the occupancy of bound water molecules near the active site indicating that a water chain is involved in the replenishment of the active site with water molecules. The structure of the novel enzyme state MCR-red1-silent at 1.8 A Ê resol- ution revealed an active site only partially occupied by coenzyme M and coenzyme B. Increased ¯exibility and distinct alternate conformations were observed near the active site and the substrate channel. The electron density of the MCR-red1-silent state aerobically co-crystallized with coenzyme M displayed a fully occupied coenzyme M-binding site with no alternate conformations. Therefore, the structure was very similar to the MCR-ox1-silent state. As a consequence, the binding of coenzyme M induced speci®c conformational changes that postulate a molecular mechanism by which the enzyme ensures that methylcoenzyme M enters the substrate channel prior to coenzyme B as required by the active-site geometry. The three different enzymatically inactive enzyme states are discussed with respect to their enzymatically active precursors and with respect to the catalytic mechanism. # 2001 Academic Press Keywords: methyl-coenzyme M reductase; methanogenesis; atomic resolution; conformational change *Corresponding author Present address: W. Grabarse, Hauptlaboratorium, BASF AG, 67065 Ludwigshafen, Germany. Abbreviations used: MCR, methyl-coenzyme reductase; MCR-ox1, MCR which exhibits the MCR-ox1 EPR signal; MCR-red1, MCR which exhibits the MCR-red1 EPR signal and which can additionally exhibit the MCR-red2 signal; MCR-silent, MCR showing no EPR signal during and prior to harvest, puri®ed from cells continuously gassed with CO 2 and H 2 (20%:80%); F 430 , factor 430, the prosthetic group of MCR. E-mail address of the corresponding author: ermler@mpibp-frankfurt.mpg.de doi:10.1006/jmbi.2001.4647 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 309, 315±330 0022-2836/01/010315±16 $35.00/0 # 2001 Academic Press