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Investigation of the kinetic mechanism and the activation of methyl -coenzyme M reductase: The catalyst for the final step in methanogenesis
Abstract
Methyl Coenzyme M Reductase (MCR) from Methanothermobacter marburgensis (Mtm) catalyzes the final step of methane formation in methanogenesis. This enzyme uses Coenzyme B (HS-CoB) as the electron donor to catalyze the two-electron reduction of methyl-Coenzyme M (CH 3-SCoM) to form methane and the heterodisulfide, CoM-SS-CoB. At the active site of MCR is cofactor F430, a nickel tetrapyrrole. Several states of MCR have been characterized. The only active form of the enzyme, called “MCRred1,” is formed by in vitro activation of the ready “MCRox1” state with a low-potential reductant, titanium(III) citrate. Based on EPR, ENDOR, and X-ray absorption spectroscopic studies and cryoreduction experiments, both the MCRox1 and MCRred1 forms of the enzyme are in the low valent Ni(I) state. Thus, the requirement of the low potential reductant titanium(III) citrate to convert Ni(I)-MCRox1 to Ni(I)-MCRred1 was puzzling. The 40 nm blue shift accompanying conversion of the ox1 to the red1 state hinted that changes in the tetrapyrrole ring might be involved in activation. Resonance Raman spectra of MCRred1, lack a vibrational band attributed to a C=N bond that is present in Ni(II)-F430, Ni(II) states of MCR, and MCRox1. These results indicate that, in the red1 state of MCR and in Ni(I)-F430, one of the conjugated C=N bonds of the tetrapyrrole ring has undergone reduction. EPR and kinetic studies of the reaction of 3-bromopropanesulfonate (BPS, I50 = 50 nM) with MCR ox1 and MCRred1 are consistent with this conclusion. Reaction of the red1 state of MCR with BPS forms a Ni(I) EPR signal called MCR BPS and propane sulfonate as a product. Reaction of the ox1 state of MCR forms an identical EPR signal without an intermediate red1 state and without forming product. Thus, the red1 state is indeed more reduced than the MCR ox1 state. These results also imply that similar redox changes may be involved in methane formation from the natural substrate, methyl-SCoM. Furthermore, we have studied the kinetic mechanism of MCR by single-turn-over kinetics. Our results indicate that Coenzyme B must react with MCR-bound methyl-Coenzyme M before even a single turnover of methane is formed.
Subject Area
Biochemistry
Recommended Citation
Horng, Yih-Chern, "Investigation of the kinetic mechanism and the activation of methyl -coenzyme M reductase: The catalyst for the final step in methanogenesis" (2003). ETD collection for University of Nebraska-Lincoln. AAI3092554.
https://digitalcommons.unl.edu/dissertations/AAI3092554