Probing the reactivity of Ni in the active site of methyl-coenzyme M reductase with substrate analogues

Abstract

Methyl-coenzyme M reductase (MCR) catalyses the reduction of methyl-coenzyme M (CH(3)-S-CoM) with coenzyme B (HS-CoB) to methane and CoM-S-S-CoB. It contains the nickel porphyrinoid F(430) as prosthetic group which has to be in the Ni(I) oxidation state for the enzyme to be active. The active enzyme exhibits an axial Ni(I)-derived EPR signal MCR-red1. We report here on experiments with methyl-coenzyme M analogues showing how they affect the activity and the MCR-red1 signal of MCR from Methanothermobacter marburgensis. Ethyl-coenzyme M was the only methyl-coenzyme M analogue tested that was used by MCR as a substrate. Ethyl-coenzyme M was reduced to ethane (apparent K(M)=20 mM; apparent V(max)=0.1 U/mg) with a catalytic efficiency of less than 1% of that of methyl-coenzyme M reduction to methane (apparent K(M)=5 mM; apparent V(max)=30 U/mg). Propyl-coenzyme M (apparent K(i)=2 mM) and allyl-coenzyme M (apparent K(i)=0.1 mM) were reversible inhibitors. 2-Bromoethanesulfonate ([I](0.5 V)=2 micro M), cyano-coenzyme M ([I](0.5 V)=0.2 mM), 3-bromopropionate ([I](0.5 V)=3 mM), seleno-coenzyme M ([I](0.5 V)=6 mM) and trifluoromethyl-coenzyme M ([I](0.5 V)=6 mM) irreversibly inhibited the enzyme. In their presence the MRC-red1 signal was quenched, indicating the oxidation of Ni(I) to Ni(II). The rate of oxidation increased over 10-fold in the presence of coenzyme B, indicating that the Ni(I) reactivity was increased in the presence of coenzyme B. Enzyme inactivated in the presence of coenzyme B showed an isotropic signal characteristic of a radical that is spin coupled with one hydrogen nucleus. The coupling was also observed in D(2)O. The signal was abolished upon exposure of the enzyme to O(2). 3-Bromopropanesulfonate ([I](0.5 V)=0.1 micro M), 3-iodopropanesulfonate ([I](0.5 V)=1 micro M), and 4-bromobutyrate also inactivated MCR. In their presence the EPR signal of MCR-red1 was converted into a Ni-based EPR signal MCR-BPS that resembles in line shape the MCR-ox1 signal. The signal was quenched by O(2). 2-Bromoethanesulfonate and 3-bromopropanesulfonate, which both rapidly reacted with Ni(I) of MRC-red1, did not react with the Ni of MCR-ox1 and MCR-BPS. The Ni-based EPR spectra of both inactive forms were not affected in the presence of high concentrations of these two potent inhibitors.

Characterization of 2-Bromoethanesulfonate as a Selective Inhibitor of the Coenzyme M-Dependent Pathway and Enzymes of Bacterial Aliphatic Epoxide Metabolism

“…A comparison of our results with the results for BES inhibition of methanogenesis revealed some interesting similarities and differences in the observed effects. For methanogens, the specific target of BES has been shown to be methyl-CoM reductase (MCR), which is inhibited in both a reversible fashion and an irreversible fashion by BES (23,24,38). MCR catalyzes the reduction of methyl-CoM to methane using a using a second thiol substrate, CoB, as shown in equation 3 (17,42,44):…”

Mechanism of Inhibition of Aliphatic Epoxide Carboxylation by the Coenzyme M Analog 2-Bromoethanesulfonate

“…The latter result is particularly interesting in that it identifies a fundamental new mechanism for inactivation of a CoM-dependent enzyme by BES. By comparison, in the case of methanogenic methyl-CoM reductase, BES inactivation occurs via oxidation of Ni(I) in the nickel tetrapyrrole cofactor F 430 to Ni(II), in the process producing a debrominated alkylsulfonate product (15). Bromopropanesulfonate, which is an even more potent inhibitor than BES, oxidizes Ni(I) in F 430 as well, but by a different mechanism (15,(25)(26)(27).…”

“…The CoM analog 2-bromoethanesulfonate (BES) was identified as a potent inhibitor of methanogenesis (12)(13)(14), and found to specifically inactivate MCR by binding as a CoM analog and rendering the enzyme inactive by oxidizing the nickel tetrapyrrole cofactor from the ϩ1 to ϩ2 oxidation state (15). Based on these studies of BES inhibition of methanogens and MCR, BES was studied as a possible inhibitor of bacterial alkene metabolism and found to specifically inhibit growth of Xanthobacter autotrophicus Py2 and Rhodococcus rhodochrous B276 with propylene, but not with carbon sources that do not use the CoM-dependent pathway and enzymes of Fig.…”

The Reaction Mechanism of Methyl-Coenzyme M Reductase

“…Several experimental and theoretical studies have been performed in an effort to understand the catalytic mechanism of MCR and the geometric and electronic structures of the intermediates in the catalytic cycle (6,10,13,16,28,30,31,33,(41)(42)(43). The major distinction between the two competing mechanisms lies in the proposed intermediate generated in the first step of catalysis.…”

“…The major distinction between the two competing mechanisms lies in the proposed intermediate generated in the first step of catalysis. The first mechanism involves formation of an organometallic methyl-Ni(III) intermediate starting from nucleophilic attack of the MCR-Ni(I) center at the carbon atom of methyl-SCoM in S N 2 fashion (6,42,43). This is followed by protonolysis to generate CH 4 and the heterodisulfide product.…”