Preparation and characterization of a (Cu,Zn)-pMMO from Methylococcus capsulatus (Bath).
We report the preparation of a (Cu,Zn)-particulate methane monooxygenase (pMMO) in which the bulk of the copper ions of the electron-transfer clusters (E-clusters) has been replaced by divalent Zn ions. The Cu and Zn contents in the (Cu,Zn)-pMMO were determined by both inductively coupled plasma mass spectroscopy (ICP-MS) and X-ray absorption K-edge spectroscopy. Further characterization of the (Cu,Zn)-pMMO was provided by pMMO-activity assays as well as low-temperature electron paramagnetic resonance (EPR) spectroscopy following reductive titration and incubation in air or air/propylene mixtures. The pMMO-activity assays indicated that the (Cu,Zn)-pMMO was no longer capable of supporting catalytic turnover of hydrocarbon substrates. However, the EPR studies revealed that the catalytic cluster (C-cluster) copper ions in the (Cu,Zn)-pMMO were still capable of supporting the activation of dioxygen when reduced, and that the 14N-superhyperfine features associated with one of the type 2 Cu(II) centers in the hydroxylation C-cluster remained unperturbed. The replacement of the E-cluster copper ions by Zn ions did compromise the ability of the protein to mediate the transfer of reducing equivalents from exogenous reductants to the C-clusters. These observations provide strong support for the electron transfer and catalytic roles for the E-cluster and C-cluster copper ions, respectively.
Chen
Chang-Li
CL
Institute of Chemistry, Academia Sinica, 128 Academia Road Section 2, Nankang, Taipei 115, Taiwan.
Chen
Kelvin H-C
KH
Ke
Shyue-Chu
SC
Yu
Steve S-F
SS
Chan
Sunney I
SI
D015502:Absorptiometry, Photon; D003300:Copper / Q000032:analysis*; D004578:Electron Spin Resonance Spectroscopy; D020619:Methylococcus capsulatus / Q000201:enzymology*; D010105:Oxygenases / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D013329:Structure-Activity Relationship; D015032:Zinc / Q000032:analysis*
D003300:Copper; D010105:Oxygenases; C021555:methane monooxygenase; D015032:Zinc