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Elucidating the process of activation of methyl-coenzyme M reductase.

ABSTRACT: Methyl-coenzyme M reductase (MCR) catalyzes the reversible reduction of methyl-coenzyme M (CH3-S-CoM) and coenzyme B (HS-CoB) to methane and heterodisulfide CoM-S-S-CoB (HDS). MCR contains the hydroporphinoid nickel complex coenzyme F430 in its active site, and the Ni center has to be in its Ni(I) valence state for the enzyme to be active. Until now, no in vitro method that fully converted the inactive MCRsilent-Ni(II) form to the active MCRred1-Ni(I) form has been described. With the potential use of recombinant MCR in the production of biofuels and the need to better understand this enzyme and its activation process, we studied its activation under nonturnover conditions and achieved full MCR activation in the presence of dithiothreitol and protein components A2, an ATP carrier, and A3a. It was found that the presence of HDS promotes the inactivation of MCRred1, which makes it essential that the activation process is isolated from the methane formation assay, which tends to result in minimal activation rates. Component A3a is a multienzyme complex that includes the mcrC gene product, an Fe-protein homolog, an iron-sulfur flavoprotein, and protein components involved in electron bifurcation. A hypothetical model for the cellular activation process of MCR is presented.

SUBMITTER: Prakash D

PROVIDER: S-EPMC4054172 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

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Elucidating the process of activation of methyl-coenzyme M reductase.

Prakash Divya D Wu Yonnie Y Suh Sang-Jin SJ Duin Evert C EC

Journal of bacteriology 20140425 13

Methyl-coenzyme M reductase (MCR) catalyzes the reversible reduction of methyl-coenzyme M (CH3-S-CoM) and coenzyme B (HS-CoB) to methane and heterodisulfide CoM-S-S-CoB (HDS). MCR contains the hydroporphinoid nickel complex coenzyme F430 in its active site, and the Ni center has to be in its Ni(I) valence state for the enzyme to be active. Until now, no in vitro method that fully converted the inactive MCRsilent-Ni(II) form to the active MCRred1-Ni(I) form has been described. With the potential ...[more]

PMID: 24769699

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Project description:Methyl-coenzyme M reductase (MCR) catalyzes the final and rate-limiting step in methane biogenesis: the reduction of methyl-coenzyme M (methyl-SCoM) by coenzyme B (CoBSH) to methane and a heterodisulfide (CoBS-SCoM). Crystallographic studies show that the active site is deeply buried within the enzyme and contains a highly reduced nickel-tetrapyrrole, coenzyme F(430). Methyl-SCoM must enter the active site prior to CoBSH, as species derived from methyl-SCoM are always observed bound to the F(430) nickel in the deepest part of the 30 A long substrate channel that leads from the protein surface to the active site. The seven-carbon mercaptoalkanoyl chain of CoBSH binds within a 16 A predominantly hydrophobic part of the channel close to F(430), with the CoBSH thiolate lying closest to the nickel at a distance of 8.8 A. It has previously been suggested that binding of CoBSH initiates catalysis by inducing a conformational change that moves methyl-SCoM closer to the nickel promoting cleavage of the C-S bond of methyl-SCoM. In order to better understand the structural role of CoBSH early in the MCR mechanism, we have determined crystal structures of MCR in complex with four different CoBSH analogues: pentanoyl, hexanoyl, octanoyl, and nonanoyl derivatives of CoBSH (CoB(5)SH, CoB(6)SH, CoB(8)SH, and CoB(9)SH, respectively). The data presented here reveal that the shorter CoB(5)SH mercaptoalkanoyl chain overlays with that of CoBSH but terminates two units short of the CoBSH thiolate position. In contrast, the mercaptoalkanoyl chain of CoB(6)SH adopts a different conformation, such that its thiolate is coincident with the position of the CoBSH thiolate. This is consistent with the observation that CoB(6)SH is a slow substrate. A labile water in the substrate channel was found to be a sensitive indicator for the presence of CoBSH and HSCoM. The longer CoB(8)SH and CoB(9)SH analogues can be accommodated in the active site through exclusion of this water. These analogues react with Ni(III)-methyl, a proposed MCR catalytic intermediate of methanogenesis. The CoB(8)SH thiolate is 2.6 A closer to the nickel than that of CoBSH, but the additional carbon of CoB(9)SH only decreases the nickel thiolate distance a further 0.3 A. Although the analogues do not induce any structural changes in the substrate channel, the thiolates appear to preferentially bind at two distinct positions in the channel, one being the previously observed CoBSH thiolate position and the other being at a hydrophobic annulus of residues that lines the channel proximal to the nickel.

Project description:Methyl-coenzyme M reductase (MCR) catalyzes the final step in methane biosynthesis by methanogenic archaea and contains a redox-active nickel tetrahydrocorphin, coenzyme F430, at its active site. Spectroscopic and computational methods have been used to study a novel form of the coenzyme, called F330, which is obtained by reducing F430 with sodium borohydride (NaBH4). F330 exhibits a prominent absorption peak at 330 nm, which is blue shifted by 100 nm relative to F430. Mass spectrometric studies demonstrate that the tetrapyrrole ring in F330 has undergone reduction, on the basis of the incorporation of protium (or deuterium), upon treatment of F430 with NaBH4 (or NaBD4). One- and two-dimensional NMR studies show that the site of reduction is the exocyclic ketone group of the tetrahydrocorphin. Resonance Raman studies indicate that elimination of this pi-bond increases the overall pi-bond order in the conjugative framework. X-ray absorption, magnetic circular dichroism, and computational results show that F330 contains low-spin Ni(II). Thus, conversion of F430 to F330 reduces the hydrocorphin ring but not the metal. Conversely, reduction of F430 with Ti(III) citrate to generate F380 (corresponding to the active MCR(red1) state) reduces the Ni(II) to Ni(I) but does not reduce the tetrapyrrole ring system, which is consistent with other studies [Piskorski, R., and Jaun, B. (2003) J. Am. Chem. Soc. 125, 13120-13125; Craft, J. L., et al. (2004) J. Biol. Inorg. Chem. 9, 77-89]. The distinct origins of the absorption band shifts associated with the formation of F330 and F380 are discussed within the framework of our computational results. These studies on the nature of the product(s) of reduction of F430 are of interest in the context of the mechanism of methane formation by MCR and in relation to the chemistry of hydroporphinoid systems in general. The spectroscopic and time-dependent DFT calculations add important insight into the electronic structure of the nickel hydrocorphinate in its Ni(II) and Ni(I) valence states.

Dataset Information

Elucidating the process of activation of methyl-coenzyme M reductase.

Publications

Elucidating the process of activation of methyl-coenzyme M reductase.

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