Here, we report a method to generate the active form of methyl-SCoM reductase (MCR) from Methanosarcina thermophila. The protocol involves adding sodium sulfide to a growing cell culture prior to harvest to yield a 'ready' (MCR(ox1)) state of the enzyme. This method can also generate a ready state of the Methanobacterium thermoautotrophicum (strain Marburg) MCR. Experiments using sodium 35S-labeled sulfide indicate the ready state that is generated involves a Ni-S adduct. As was shown earlier for the Mb. thermoautotrophicum MCR(ox1) [Goubeaud, M., Schreiner, G. and Thauer, R. K. (1997) Eur. J. Biochem. 17, 2374-2377], this ready state is converted to the highly active MCR(red1) form by reductive activation with Ti(III) citrate. The reduction of MCR(ox1) to MCR(red1) with concomitant increase in activity demonstrated that MCR(red1) is the active form of MCR from Ms. thermophila. We also observed the loss of the 35S-sulfide label from the enzyme when MCR(ox1) was converted to MCR(red1). Other states of MCR could be generated in the whole cells by adding different potential ligands to the cell medium; for example, the MCR(ox2) state was generated by treating cells with sodium sulfite or sodium dithionite.
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