Investigation of Large Protein Complexes Important for Methyl-Coenzyme M Reductase Activity and Function

Abstract

Methyl-coenzyme M reductase (Mcr) is a key enzyme of methanogens that catalyzes the production or consumption of methane. Mcr contains a nickel tetrapyrrole cofactor (coenzyme F430) which has a crucial role in catalysis. The enzyme is only active when its cofactor is in the Ni(I) state, and it is extremely oxygen sensitive. Therefore, the activity is quickly lost without special precautions. Until now, the details on how Mcr gets activated intracellularly are not clear. Here we try to get a better understanding of the activation process. In vivo activation of Mcr involves two components, A2 and an iron-sulfur-containing large complex named A3a. In Dr. Duin's laboratory, native A3a from Methanothermobacter marburgensis was purified and its subunits were characterized. It was proposed that there is a core of activating proteins that include McrC, iron-sulfur flavoprotein (FFP), Mmp7 (methanogenesis marker protein 7), component A2, and ATP binding protein. Characterization of these proteins and pull-down experiments should provide a more complete picture of the actual activation complex and how it works. Genes from Methanococcus maripaludis were cloned in a pMEV4mTs plasmid with a strep Flag-Strep2 tag and successfully expressed homologously in M. maripaludis. Recombinant proteins were purified with Strep-Tactin Superflow Plus resin. As expected, additional protein bands were detected on SDS-PAGE. The identity of these bands for recombinant Mmp7, A2 components, and McrC was verified by MALDI mass spectrometry. It is assumed that the additional bands are probably due to the direct interactions of recombinant protein with the other proteins involved in the Mcr activation. McrC with strep tag made a complex with Mmp7, Mmp3, Mmp17, and Mcr enzyme. The A2 component interacts with the Mcr. Mmp7 with strep tag made a complex with McrA, Mmp3, and McrB. Anaerobic purified recombinant Mmp7 has a dark brownish color. EPR and UV–visible results showed that it contains an iron-sulfur cluster. The same was true for the A2 protein. FFP has a strong yellow color. The UV–visible and EPR results showed that a flavin and an iron-sulfur cluster are present in the sample. Methanogenesis marker proteins are signature proteins of the archaea. In the Methanosarcina acetivorans C2 genome, there is an operon named mmp operon, which contains A2 (atwA), mmp3, mmp6, mmp5, mmp15, mmp17, and mmp7. It is assumed that these proteins form one complex and be involved in the activation and folding of Mcr. All genes were cloned in one pETDuet vector and expressed simultaneously in C41 (E. coli) cells. Since the A2 component can bind ATP, the A2 component was purified directly from cell extract with an ATP agarose column. Mass spectrometry results showed that except for Mmp3, all 7 proteins came down in one complex. Mmp3 is present, however, when the complex is isolated under the exclusion of air.