Architecture and active site of particulate methane monooxygenase

Abstract

Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria, organisms that live on methane gas as their sole carbon source. Understanding pMMO function has important implications for bioremediation applications and for the development of new, environmentally friendly catalysts for the direct conversion of methane to methanol. Crystal structures of pMMOs from three different methanotrophs reveal a trimeric architecture, consisting of three copies each of the pmoB, pmoA, and pmoC subunits. There are three distinct metal centers in each protomer of the trimer, mononuclear and dinuclear copper sites in the periplasmic regions of pmoB and a mononuclear site within the membrane that can be occupied by copper or zinc. Various models for the pMMO active site have been proposed within these structural constraints, including dicopper, tricopper, and diiron centers. Biochemical and spectroscopic data on pMMO and recombinant soluble fragments, denoted spmoB proteins, indicate that the active site involves copper and is located at the site of the dicopper center in the pmoB subunit. Initial spectroscopic evidence for O(2) binding at this site has been obtained. Despite these findings, questions remain about the active site identity and nuclearity and will be the focus of future studies.

Figure 1

Overall architecture of pMMO. (A) Structure of Methylocystis sp. strain M pMMO protomer (PDB accession code 3RGR). The pmoB, pmoA, and pmoC subunits are shown in blue, magenta, and green, respectively. The N- and C-termini of pmoB are labeled. An exogenous helix is shown in yellow. Copper ions are shown as cyan spheres and a zinc ion is shown as a gray sphere. Ligands are shown as ball-and-stick representations. (B) Structure of M. capsulatus (Bath) pMMO protomer (PDB accession code 3RGB). The amino terminal domain of pmoB (spmoBd1) is shown in blue, the carboxy terminal domain of pmoB is shown in gray (spmoBd2), and the two transmembrane helices are shown in transparent blue. In the recombinant spmoB protein, spmoBd1 and spmoBd2 are linked by a GKLGGG sequence connecting residues 172 and 265 (labeled). The pmoA and pmoC subunits are shown in transparent magenta and transparent green, respectively. A hydrophilic patch of residues proposed to house a tricopper active site is denoted with an asterisk. The mononuclear copper site at the interface of the two spmoB domains is not present in the Methylocystis sp. strain M pMMO structure. [A color version of this figure is available online.]

Figure 2

Metal centers modeled in the pMMO crystal structures. The pmoB site modeled as dicopper in the M. capsulatus (Bath) pMMO structure is modeled as dicopper in one protomer and monocopper in two protomers in the Methylocystis sp. strain M pMMO structure. The monocopper site in pmoB from M. capsulatus (Bath) pMMO does not contain copper in the other pMMO structures. In both structures, zinc is found in the transmembrane site as a result of including zinc in the crystallization buffer. Copper occupies this site in M. trichosporium OB3b pMMO. [A color version of this figure is available online.]

Figure 3

Multiple sequence alignments of representative pmoB/amoB and pmoC/amoC sequences containing the ligands to the crystallographically observed pMMO metal centers. The ligands to the pmoB dicopper site are shown in magenta, the ligands to the pmoB monocopper site in M. capsulatus (Bath) pMMO are shown in blue, and the ligands to the zinc site are shown in green. The ligands to the dicopper center are not conserved in the bottom four Verrucomicrobia sequences. [A color version of this figure is available online.]

Figure 4

Optical spectrum of detergent-solubilized pMMO from M. capsulatus (Bath) (top) and difference spectra of recombinant spmoB (bottom) reduced and oxidized with either H₂O₂ or O₂. The feature at 345 nm is similar to that observed for the μ-η²:η²-peroxo Cu^II₂ species formed in hemocyanin and tyrosinase. The peak at 410 nm derives from heme contaminants in the pMMO sample.