Enzymatic oxidation of methane

Abstract

Methane monooxygenases (MMOs) are enzymes that catalyze the oxidation of methane to methanol in methanotrophic bacteria. As potential targets for new gas-to-liquid methane bioconversion processes, MMOs have attracted intense attention in recent years. There are two distinct types of MMO, a soluble, cytoplasmic MMO (sMMO) and a membrane-bound, particulate MMO (pMMO). Both oxidize methane at metal centers within a complex, multisubunit scaffold, but the structures, active sites, and chemical mechanisms are completely different. This Current Topic review article focuses on the overall architectures, active site structures, substrate reactivities, protein-protein interactions, and chemical mechanisms of both MMOs, with an emphasis on fundamental aspects. In addition, recent advances, including new details of interactions between the sMMO components, characterization of sMMO intermediates, and progress toward understanding the pMMO metal centers are highlighted. The work summarized here provides a guide for those interested in exploiting MMOs for biotechnological applications.

Figure 1

sMMO and pMMO operons.

Figure 2

Overall architecture of MMOs. (A) The sMMO hydroxylase (MMOH, PDB accession code 1MTY) with α subunits shown in gray, β subunits shown in teal, and γ subunits shown in wheat. Each α₂β₂γ₂ dimer contains two diiron active sites (orange spheres). (B) The pMMO trimer (PDB accession code 3RGB) with pmoB subunits shown in gray, pmoA subunits shown in teal, and pmoC subunits shown in wheat. Copper ions are shown as cyan spheres and zinc ions are shown as gray spheres.

Figure 3

One αβγ protomer of Methylocystis sp. strain Rockwell pMMO (PDB accession code 4PHZ) showing the unidentified helix (purple) and two bound lipids (green and yellow). The unidentified helix is also observed in structures of pMMO from M. trichosporium OB3b and Methylocystis sp. strain M. The lipid shown in green forms hydrogen bonds with conserved arginine residues from pmoC and mediates the interaction between pmoC and the mystery helix.

Figure 4

Active site of sMMO. (A) Oxidized diiron center (PDB accession code 1MTY). (B) Reduced diiron center (PDB accession code 1FYZ). Iron ions are shown as orange spheres and solvent ligands are shown as red spheres.

Figure 5

Metal centers in the pMMO crystal structures. The locations of the dicopper, monocopper, and zinc/copper sites are shown as cyan and gray spheres within a protomer of M. capsulatus (Bath) pMMO (left). In the spmoB protein, two transmembrane helices (gray) linking the periplasmic N- and C-terminal cupredoxin domains of pmoB (magenta) are replaced with a GKLGGG linker connecting residue 172 to residue 265. The structure of spmoB has not been determined. The M. capsulatus (Bath) pMMO structure contains dicopper, monocopper, and zinc sites (top right, PDB accession code 3RGB). In the Methylocystis sp. strain Rockwell pMMO structure (bottom right), the dicopper site is modeled with a single copper ion and a solvent molecule, and the zinc/copper site is occupied by copper (PDB accession code 4PHZ). Soaking the crystals in zinc reveals a fourth ligand to this site, Glu201 (PDB accession code 4PI2). Solvent ligands are shown as red spheres.

Figure 6

Substrate binding by MMOH. (A) Three cavities and a pore region connect to the active site: cavity 1, green; cavity 2, wheat; cavity 3, light blue; hydrophilic pore, magenta. The iron ions are shown as orange spheres. (B) Structure of active site with bound 2-bromoethanol (PDB accession code 1XVG). The carbon atoms of 2-bromoethanol are shown in green and the bromine atom is shown in magenta.

Figure 7

Structural changes in MMOH upon complexation with MMOB. (A) MMOB (purple cartoon) binds in the canyon region formed by the two MMOH protomers (black and gray surfaces) (PDB accession code 4GAM). (B) Active site region in uncomplexed MMOH (PDB accession code 1MTY). The hydrophilic pore is open to solvent, and cavities 1 and 2 are separated by Leu110 and Phe188. (C) Active site region in MMOH-MMOB complex (PDB accession code 4GAM). The pore is closed, and conformational changes in Phe188 link cavities 1 and 2, allowing substrate access to the active site. Cavities are shown as green mesh.