Copper Ions and The Particulate Methane Monooxygenase from Methylococcus capsulatus (Bath)

• Main Authors: Chang-Li Chen, 陳昌立
• Other Authors: Sunney I. Chan
• Format: Others
• Language: en_US
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/bk48ja

博士 === 國立清華大學 === 化學系 === 92 === Copper ions play an essential role in particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath). The particulate methane monooxygenase (pMMO) contains 15 reduced copper ions which are arranged in five trinuclear clusters. Two of these clusters were subsequently found to participate in dioxygen chemistry and hydrocarbon hydroxylation chemistry, called C-clusters. The remaining copper ions were in the reduced d10 state, and were thought to be responsible for channeling electrons to the C-clusters from NADH, called E-clusters. The low temperature EPR spectrum of as-isolated pMMO was deconvoluted into a type 2 Cu(II) signal and a broad, but nearly isotropic EPR signal centered at g ~ 2.1. Earlier magnetization and magnetic susceptibility measurements have suggested that the latter EPR signal, which is not sensitive to microwave power saturation, arise from a ferromagnetically exchange-coupled trinuclear Cu(II) cluster with J = 15�{20 cm–1 and with a zero-field splitting D of +0.018cm-1 (175 G) and E value of 0.005 cm-1 (50 G). By combining EPR spectroscopy and rapid cryogenically trap, we successfully observed the different oxidative phases of the turnover cycle and practically proved the catalytic mechanisms of pMMO. Processing cell growth in a fermentor adapted with a hollow-fiber bioreactor, we successfully prepared the (Cu, Zn)-pMMO. The bulk of the copper ions of the E-clusters have been replaced by divalent Zn ions in (Cu, Zn)-pMMO. The Cu and Zn contents in the (Zn, Cu)-pMMO were determined by both ICP-MS and x-ray absorption K-edge spectroscopy. Further characterization of the (Zn, Cu)-pMMO was provided by low temperature electron paramagnetic spectroscopy during reductive titration and hydrocarbon hydroxylation. These studies indicate that the (Zn, Cu)-pMMO is still capable of supporting the activation of dioxygen, but that the replacement of the E-cluster copper ions has compromised the ability of the protein to mediate the transfer of reducing equivalents to the C-clusters. These observations provide strong support for the electron transfer and catalytic roles that we have previously proposed for the E-cluster and C-cluster copper ions, respectively.