ACTIVATION OF METHYL-SCOM REDUCTASE TO HIGH SPECIFIC ACTIVITY AFTER TREATMENT OF WHOLE CELLS WITH SODIUM SULFIDE

Here, we report a method to generate the active form of methyl-SCoM re ductase (MCR) from Methanosarcina thermophila. The protocol involves a dding sodium sulfide to a growing cell culture prior to harvest to yie ld a ''ready'' (MCRox1) state of the enzyme, This method can also gene rate a ready state of the Methanobacterium thermoautotrophicum (strain Marburg) MCR. Experiments using sodium S-35-labeled sulfide indicate the ready state that is generated involves a Ni-S adduct. As was shown earlier for the Mb. thermoautotrophicum MCRox1 [Goubeaud, M., Schrein er, G. and Thauer, R. K. (1997) Eur. J. Biochem. 17, 2374-2377], this ready state is converted to the highly active MCRred1 form by reductiv e activation with Ti(III) citrate, The reduction of MCRox1 to MCRred1 with concomitant increase in activity demonstrated that MCRred1 is the active form of MCR from Ms. thermophila. We also observed the loss of the S-35-sulfide label from the enzyme when MCRox1 was converted to M CRred1 Other states of MCR could be generated in the whole cells by ad ding different potential ligands to the cell medium; for example, the MCRox2 state was generated by treating cells with sodium sulfite or so dium dithionite.