Structural Analysis of a Ni-Methyl Species in Methyl-Coenzyme M Reductase from Methanothermobacter marburgensis

Abstract

We present the 1.2 Å resolution X-ray crystal structure of a Ni-methyl species that is a proposed catalytic intermediate in methyl-coenzyme M reductase (MCR); the enzyme that catalyzes the biological formation of methane. The methyl group is situated 2.1 Å proximal of the Ni-atom of the MCR coenzyme F430. A rearrangement of the substrate channel has been posited to bring together substrate species, but Ni(III)-methyl formation alone does not lead to any observable structural changes in the channel.

The Reaction Mechanism of Methyl-Coenzyme M Reductase

“…The three subunits (␣␤␥) tightly associate to form two 50-Å hydrophobic channels (one in each heterotrimer) (6) ending in a pocket that accommodates a redox-sensitive nickel tetrapyrrole cofactor (coenzyme F430), which plays an essential role in catalysis (7,8). Currently, 16 distinct enzymatic and complexed states of MCR have been spectroscopically characterized, including EPR-active (MCR red1 (Ni(I)-F430), MCR red2 (rhombic Ni(I)-F430), MCR ox1 (high spin Ni(II) thiyl-radical), MCR ox2 (high spin rhombic Ni(II) thiyl-radical), MCR Me (methyl-Ni(III)), and related alkyl-Ni(III)) (9)(10)(11)(12)(13)(14) and MCR silent (meaning that the enzyme is in an EPR-silent Ni(II) form, which includes MCR red1-silent (five coordinate Ni(II)-F430), and MCR ox1-silent (Ni(II) thiolate) (6,15,16). To initiate catalysis, the enzyme must be in the MCR red1 state, which contains the redox-active nickel as Ni(I) (7,8).…”

“…Although x-ray structures are only available from one Ni(III) (methyl-Ni) and inactive Ni(II) states, all of them show that both substrates access the active site through the same long narrow channel, which opens into the hydrophobic cavity above the hydrocorphinoid plane of F430 (6,10,15,16,19). The phosphate group of CoB 7 SH is positioned by ionic interactions with MCR residues located halfway down the channel with its thiol group located 8.7 Å from the nickel.…”

“…Mechanism I involves attack of the Ni(I) nucleophile on the methyl group of methyl-SCoM to generate a methyl-Ni(III) intermediate (16,26). This proposed mechanism I is based on mechanistic work with F430 model complexes (27), on the location of substrates in the active site of inactive Ni(II) MCR structures (6), and on mechanistic and crystallographic studies of the active Ni(I) enzyme with 3-bromopropanesulfonate and methyl halide (10,11,13,28). Mechanism II starts with Ni(I) attack on the sulfur atom of methylSCoM, promoting the homolytic cleavage of the methyl-sulfur bond and generating a methyl radical ( ⅐ CH 3 ) and a Ni(II)⅐thiolate complex.…”

“…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.…”

“…This is followed by protonolysis to generate CH 4 and the heterodisulfide product. The putative methyl-Ni(III) intermediate has been generated independently by adding methyl halides to the active MCRNi(I) and is characterized by EPR spectroscopy (13,28) and x-ray crystal structure (10). The second mechanism involves a direct attack of the Ni(I) on the S of methyl-SCoM, resulting in a homolytic cleavage of the thioether bond and formation of a Ni(II)-thiolate species and a ⅐ CH 3 .…”