Product analog binding identifies the copper active site of particulate methane monooxygenase

Abstract

Nature's primary methane-oxidizing enzyme, the membrane-bound particulate methane monooxygenase (pMMO), catalyzes the oxidation of methane to methanol. pMMO activity requires copper, and decades of structural and spectroscopic studies have sought to identify the active site among three candidates: the Cu_B, Cu_C, and Cu_D sites. Challenges associated with the isolation of active pMMO have hindered progress toward locating its catalytic center. However, reconstituting pMMO into native lipid nanodiscs stabilizes its structure and recovers its activity. Here, these active samples were incubated with 2,2,2,-trifluoroethanol (TFE), a product analog that serves as a readily visualized active-site probe. Interactions of TFE with the Cu_D site were observed by both pulsed ENDOR spectroscopy and cryoEM, implicating Cu_D and the surrounding hydrophobic pocket as the likely site of methane oxidation. Use of these orthogonal techniques on parallel samples is a powerful approach that can circumvent difficulties in interpreting metalloenzyme cryoEM maps.

Fig. 1.. CryoEM structure of M. capsulatus (Bath) pMMO in native lipid nanodiscs.

a, The α₃β₃γ₃ C3 symmetrical trimer with one protomer highlighted (PDB accession code 7S4H). The PmoB, PmoA, and PmoC subunits are shown in purple, teal, and gold, respectively, with the copper ions as cyan spheres. b, The bis-His site. c, The Cu_B site. d, The neighboring Cu_C and Cu_D sites. The Cu_C site is unoccupied, and the Cu_D site is modeled as copper.

Fig. 2.. Parallel EPR and cryoEM studies of pMMO in native lipid nanodiscs.

a, X-band CW EPR spectra of pMMO (black), metal-depleted pMMO (pink), and copper-reloaded pMMO (teal). The top bracket represents the ${\mathrm{A}}_{\Vert }$ hyperfine splitting in the signal attributed to the Cu_B site, and the bottom bracket represents the ${\mathrm{A}}_{\Vert }$ hyperfine splitting attributed to a secondary Cu(II) species, shown in this report to arise from Cu_D. X-band EPR conditions: 9.371 (top), 9.368 (middle), 9.365 (bottom) GHz, 30 K, 12.5 G modulation, 250 μW microwave power, 320 ms time constant, 10 G/s scan rate, 3 scans. b, Methane oxidation activity for as-isolated pMMO, metal-depleted pMMO, and copper-reloaded pMMO in native lipid nanodiscs. c, CryoEM map and model for one protomer of metal-depleted pMMO (3.22 Å resolution) and its Cu_B site showing attenuated density for the copper ion. The PmoC subunit is disrupted in this structure, and the ligands for the Cu_C and Cu_D sites cannot be modeled in the density. d, CryoEM map and model for one protomer of copper-reloaded pMMO (3.12 Å resolution) and its Cu_B site showing recovered density for the copper ion. Maps are shown with local resolution coloring (blue 2.5 Å, white 3.0 Å, red 3.5 Å). e, CryoEM density at the Cu_C and Cu_D sites in copper-reloaded pMMO.

Fig. 3.. ENDOR spectroscopic analysis of TFE interacting with the pMMO copper sites in native lipid nanodiscs.

a-c, Q-band ¹H/¹⁹F Mims pulsed ENDOR of pMMO with (blue) and without (gold) the addition of 20x TFE at (a) ${\mathrm{g}}_{\Vert }=2.14$ (~11200 G), (b) $g_{\perp }=2.05$ (~12050 G), (c) low-field edge of EPR spectrum, g = 2.32 (~10650 G). Mims conditions: microwave (MW) frequency = 34.72 GHz (with) and 34.59 GHz (without), 2 K, $\pi ∕2=50$ ns, $\tau =900$ ns, ${\mathrm{T}}_{\text{RF}}=60$ μs, RF tail = 5 μs, repetition time of 100 ms, ~20-50 scans each. For description of the sample solutions, see Methods. d, Model derived from the ¹⁹F ENDOR data.

Fig. 4.. ENDOR detection of a specific interaction of the pMMO Cu_D site with TFE in native lipid nanodiscs.

Q-band ¹H/¹⁹F Mims pulsed ENDOR of pMMO a, plus 20x TFE; b, without fluorocarbon addition; c, reduced with ascorbate plus 20x TFE; d, solubilized in DDM plus 20x TFE; e, plus 20x 4-fluorophenol. All spectra were acquired at $g_{\Vert }$ ($g=2.16$). Mims conditions: MW frequency = 34.59 – 34.74, 2 K, $\pi ∕2=50$ ns, $\tau =900$ ns, TRF = 60 μs, RF tail = 15 μs, repetition time of 100 ms, ~20 scans each.

Fig. 5.. CryoEM structures of pMMO in native lipid nanodiscs with 0x TFE (gold) and 20x TFE (blue).

a, The bis-His site. b, The Cu_B site. In the 20x-TFE structure, density for the posterior axial water appears to connect to the backbone amino group of His137, while the anterior axial water shows weaker density. c, The Cu_C and Cu_D sites in the 0x-TFE and 20x-TFE structures. In the 20x-TFE structure, an oblong density modeled as TFE connects to the Cu_D site. d, Omit map (F_O – F_C) for the 0x-TFE structure in which two water molecules were removed from the model. e, Omit map (F_O – F_C) for the 20x-TFE structure with TFE omitted from the model. f, Difference map (F_O¹ – F_O²) showing the difference between the 20x-TFE and 0x-TFE cryoEM maps at $6\sigma$. g, Hydrophobic cavity housing TFE.