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 reductase (MCR) from Methanosarcina thermophila. The protocol involves adding sodium sulfide to a growing cell culture prior to harvest to yield a "ready" (MCRox1) 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 MCRox1 [Goubeaud, M., Schreiner, G. and Thauer, R. K. (1997) Eur. J. Biochem. 17, 2374-2377], this ready state is converted to the highly active MCRred1 form by reductive 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 35S-sulfide label from the enzyme when MCRox1 was converted to MCRred1. Other states of MCR could be generated in the whole cells by adding different potential ligands to the cell medium; for example, the MCRox2 state was generated by treating cells with sodium sulfite or sodium dithionite.
Becker
D F
DF
Department of Biochemistry, Beadle Center, University of Nebraska, Lincoln, Nebraska 68588-0664, USA.
Ragsdale
S W
SW
D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D007700:Kinetics; D017014:Methanobacterium / Q000187:drug effects / Q000201:enzymology; D017020:Methanosarcina / Q000187:drug effects / Q000201:enzymology / Q000254:growth & development; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D013053:Spectrophotometry; D013440:Sulfides / Q000494:pharmacology
D013440:Sulfides; D010088:Oxidoreductases; C021572:methyl coenzyme M reductase; C033479:sodium sulfide