Evidence for a Metal−Thiolate Intermediate in Alkyl Group Transfer from Epoxypropane to Coenzyme M and Cooperative Metal Ion Binding in Epoxyalkane:CoM Transferase

Abstract

Epoxyalkane:coenzyme M transferase (EaCoMT) catalyzes the nucleophilic addition of coenzyme M (CoM, 2-mercaptoethanesulfonic acid) to epoxypropane forming 2-hydroxypropyl-CoM. The biochemical properties of EaCoMT suggest that the enzyme belongs to the family of alkyltransferase enzymes for which Zn plays a role in activating an organic thiol substrate for nucleophilic attack on an alkyl-donating substrate. The enzyme has a hexameric (alpha(6)) structure with one zinc atom per subunit. In the present work M(2+) binding and the role of Zn(2+) in EaCoMT have been established through a combination of biochemical, calorimetric, and spectroscopic techniques. A variety of metal ions, including Zn(2+), Co(2+), Cd(2+), and Ni(2+), were capable of activating a Zn-deficient "apo" form of EaCoMT, affording enzymes with various levels of activity. Titration of Co(2+) into apo-EaCoMT resulted in UV-visible spectroscopic changes consistent with the formation of a tetrahedral Co(2+) binding site, with coordination of bound Co(2+) to two thiolate ligands. Quantification of UV-visible spectral changes upon Co(2+) titration into apo-EaCoMT demonstrated that EaCoMT binds Co(2+) cooperatively at six interacting sites. Isothermal titration calorimetric studies of Co(2+) and Zn(2+) binding to EaCoMT also showed cooperativity for metal ion binding among six sites. The addition of CoM to Co(2+)-substituted EaCoMT resulted in UV-visible spectral changes indicative of formation of a new thiol-Co(2+) bond. Co(2+)-substituted EaCoMT exhibited a unique Co(2+) EPR spectrum, and this spectrum was perturbed significantly upon addition of CoM. The presence of a divalent metal ion was required for the release of protons from CoM upon binding to EaCoMT, with Zn(2+), Co(2+), and Cd(2+) each facilitating proton release. The divalent metal ion of EaCoMT is proposed to play a key role in the coordination and deprotonation of CoM, possibly through formation of a metal-thiolate that is activated for attack on the epoxide substrate.

Getting a Handle on the Role of Coenzyme M in Alkene Metabolism

“…In methanogenic methyltransferases, zinc plays an integral role in activation of the thiol group of CoM for acceptance of a methyl group from various donors (22,55). Analogously, the zinc atom in EaCoMT functions in the activation of CoM as well, in this instance for attack on the electrophilic epoxide substrate (9). In both methanogenesis and epoxide carboxylation, reductive dealkylation of a CoM thioether occurs, in the former system to generate methane and in the latter to form an enolate that undergoes carboxylation (11,48,50).…”

“…The EaCoMT from Xanthobacter has been the subject of intensive characterization (4,9,37). Xanthobacter EaCoMT is a hexameric protein containing 1 Zn atom per subunit and is highly specific for CoM as the organic thiol substrate (37).…”

“…EaCoMT, like MetE, is believed to coordinate the substrate thiol at a zinc center, thereby lowering the pK a of the thiol such that it can be deprotonated and serve as a nucleophile for attack on the second substrate, epoxypropane (9). For MetE, extended X-ray absorption fine structure analysis has shown that the zinc environment changes from a 2S ϩ 2N/O environment to a 3S ϩ 1N/O environment upon binding homocysteine (23,52,72).…”

Integrated Paramagnetic Resonance of High-Spin Co(II) in Axial Symmetry: Chemical Separation of Dipolar and Contact Electron−Nuclear Couplings

“…1 The cobalt ion typically adopts a high-spin (hs) configuration and a coordination geometry similar to that of the native zinc complex. In contrast to Zn(II), which is only accessible by X-ray absorption spectroscopy (XAS), [2][3][4][5][6][7][8][9] complexes of hs Co(II), including those of enzymes, are amenable to a wide array of spectroscopies, including optical spectroscopy, XAS, [10][11][12][13][14][15][16][17] magnetic circular dichroism (MCD), [18][19][20][21][22][23][24] (EPR), 15,16,21,[25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44] NMR and, more recently, electronnuclear double resonance (ENDOR) 69 and high-frequency/ fi...…”

A Cysteine-Rich CCG Domain Contains a Novel [4Fe-4S] Cluster Binding Motif As Deduced from Studies with Subunit B of Heterodisulfide Reductase from Methanothermobacter marburgensis

“…The primary sequence of HdrA indicates that it contains the FAD binding site and four canonical binding motifs for [4Fe-4S] clusters. HdrB contains no sequence motif characteristic for the binding of known cofactors but has two unique cysteine-rich sequence motifs (CX [31][32][33][34][35][36][37][38][39] CCX [35][36] CXXC) of unknown function, designated as the CCG domain in the Pfam protein families database (accession number PF02754) (8). The ferredoxin-like subunit HdrC contains two canonical binding motifs for [4Fe-4S] clusters (9).…”