The metal centers of particulate methane monooxygenase from Methylosinus trichosporium OB3b

Abstract

Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. The nature of the pMMO active site and the overall metal content are controversial, with spectroscopic and crystallographic data suggesting the presence of a mononuclear copper center, a dinuclear copper center, a trinuclear center, and a diiron center or combinations thereof. Most studies have focused on pMMO from Methylococcus capsulatus (Bath). pMMO from a second organism, Methylosinus trichosporium OB3b, has been purified and characterized by spectroscopic and crystallographic methods. Purified M. trichosporium OB3b pMMO contains approximately 2 copper ions per 100 kDa protomer. Electron paramagnetic resonance (EPR) spectroscopic parameters indicate that type 2 Cu(II) is present as two distinct species. Extended X-ray absorption fine structure (EXAFS) data are best fit with oxygen/nitrogen ligands and reveal a Cu-Cu interaction at 2.52 A. Correspondingly, X-ray crystallography of M. trichosporium OB3b pMMO shows a dinuclear copper center, similar to that observed previously in the crystal structure of M. capsulatus (Bath) pMMO. There are, however, significant differences between the pMMO structures from the two organisms. A mononuclear copper center present in M. capsulatus (Bath) pMMO is absent in M. trichosporium OB3b pMMO, whereas a metal center occupied by zinc in the M. capsulatus (Bath) pMMO structure is occupied by copper in M. trichosporium OB3b pMMO. These findings extend previous work on pMMO from M. capsulatus (Bath) and provide new insight into the functional importance of the different metal centers.

Figure 1

SDS–PAGE gel of purified M. trichosporium OB3b pMMO (30 μg). Molecular mass standards and pMMO subunits are labeled.

Figure 2

X-band EPR spectra of membrane-bound (A), solubilized (B), and purified (C) M. trichosporium OB3b pMMO. The spectra have been normalized for easier comparison. Experimental conditions: temperature, 77 K; microwave frequency, 9.21 GHz; microwave power, 7 mW; 100 kHz field modulation amplitude, 5 G; time constant, 300 ms; scan time, 4 min.

Figure 3

X-band EPR of purified M. trichosporium OB3b pMMO. (A) The experimental spectrum. (B) The simulation of the major component with parameters: g_⊥= 2.052, g_|| = 2.247, A(⁶³Cu)_||= 585 MHz, A(⁶³Cu)_⊥ = 60 MHz (this last value has significant uncertainty); Gaussian line shapes are used with single-crystal line widths of 100 MHz (half-width at half-maximum). (C) The simulation of the minor component with parameters: g_⊥= 2.060, g_|| = 2.225, A(⁶³Cu)_|| = 585 MHz, A(⁶³Cu)_⊥= 60 MHz (both ⁶³Cu hyperfine couplings are set equal to the values determined for the major component) and with hyperfine couplings to two equivalent nitrogen nuclei: A(¹⁴N)isotropic = 40 MHz; line widths are 18 MHz at g_⊥and 40 MHz at g_||. The relative intensities of the two have been scaled to approximate their relative amounts, but precise quantitation is not implied.

Figure 4

Copper XANES spectrum of purified M. trichosporium OB3b pMMO. The solid vertical line at 8984 eV identifies spectral features corresponding to the Cu(I) 1s → 4p transition, and the inset shows an expanded view of the Cu(II) 1s → 3d transition.

Figure 5

Copper EXAFS fitting analysis for purified M. trichosporium OB3b pMMO. (A) Raw unfiltered EXAFS data (black) and simulations (gray) for copper bound to pMMO. (B) Fourier transforms of the raw EXAFS (black) and best-fit simulation (gray).

Figure 6

Crystal structure of M. trichosporium OB3b pMMO protomer with pmoB shown in pink, pmoA in light blue, and pmoC in light green. Modeled copper ions are shown as cyan spheres. Two helices in pmoC that differ from the M. capsulatus (Bath) structure are shown in yellow. The helix in front, which is not present in the M. capsulatus (Bath) structure, is shown with the 2F_o − F_c electron density superimposed at 1σ. The direction of the yellow helix in back has been reversed with respect to the M. capsulatus (Bath) pMMO structure.

Figure 7

Copper anomalous difference Fourier maps of the three metal centers in M. trichosporium OB3b pMMO. Left: The location of the monocopper center in M. capsulatus (Bath) pMMO. No anomalous density is observed. Middle: The location of the dicopper center in M. capsulatus (Bath) pMMO with the anomalous map contoured at 4σ. Right: The location of the “zinc” center M. capsulatus (Bath) pMMO with the anomalous map contoured at 3σ. Side chain positions are not well defined at this resolution, and details of coordination cannot be acsertained. Oxygen atoms are colored red, nitrogen atoms are colored blue, and carbon atoms are colored according to subunit as in Figure 6.