Feature Article The Copper Clusters in the Particulate Methane Monooxygenase (pMMO) from Methylococcus capsulatus (Bath) Kelvin H.-C. Chen a,b ( ), Chang-Li Chen a,b ( ), Chiu-Fou Tseng a,b ( ), Steve S.-F. Yu a ( ), Shyue-Chu Ke a,d ( ), Jyh-Fu Lee c ( ), Hiephoa T. Nguyen, f Sean J. Elliott, f James O. Alben e and Sunney I. Chan a,b,f *( ) a Institute of Chemistry, Academia Sinica, Nankang, Taipei 115, Taiwan, R.O.C. b Department of Chemistry, National Tsing Hua University, Hsinchu 300, Taiwan, R.O.C. c National Synchrotron Radiation Research Center, Hsinchu 300, Taiwan, R.O.C. d Department of Physics, National Dong Hwa University, Hualien, Taiwan, R.O.C. e Department of Medical Biochemistry, The Ohio State University, Columbus, Ohio 43210, U.S.A. f A.A. Noyes Laboratory of Chemical Physics, 127-72 California Institute of Technology, Pasadena, California 91125, U.S.A. The particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) is a multi- copper protein that hydroxylates methane and other small n-alkanes to their corresponding alcohols with high regiospecificity and stereoselectivity. The copper ions appear to be arranged into ~5 trinuclear copper clus- ters. Two of these clusters are thought to be involved in the dioxygen chemistry and alkane hydroxylation me- diated by the enzyme. Accordingly, these catalytic clusters (C-clusters) are typically oxidized, as the protein is isolated. The remaining copper ions are normally reduced, and it has been suggested that they provide a res- ervoir of reducing equivalents needed for the turnover of the enzyme. In this study, we have oxidized the pro- tein to different levels of oxidation of the copper ions using dioxygen, hydrogen peroxide, ferricyanide, and dioxygen in the presence of the suicide substrate acetylene, and have characterized the oxidized copper cen- ters using low temperature electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy. The results are consistent with the grouping of the copper ions into catalytic clusters and electron transfer clusters (E-clusters). Quantification indicates that there are, indeed, two C-clusters. A model in which the two C-clusters are activated by dioxygen starting from their fully reduced states and mediate the hydroxylation chemistry of methane will be presented and discussed. Keywords: Copper clusters; pMMO; Redox titrations; Suicide substrate; Low temperature EPR; X-ray absorption spectroscopy. INTRODUCTION Methanotrophic bacteria utilize methane both as a car- bon and an energy source. 1 The oxidation of methane by aero- bic methanotrophs is mediated by the methane monooxy- genases (MMOs), the classical monooxygenases that utilize two reducing equivalents to split the O-O bond of dioxygen and transfer one of the oxygen atoms to a hydrocarbon sub- strate. In pMMO, the latter oxygen atom is incorporated into the C-H bond of methane to form methanol, while the other is reduced to form H2 O. The controlled conversion of methane to methanol in the chemical laboratory is extremely difficult to realize and requires the use of expensive catalysts in addi- tion to high temperatures and pressures. 2 In contrast, the bio- logical process in Nature is quite facile even under ambient temperatures and pressures. There are two forms of the MMOs in methanotrophic bacteria. One form is found in the cytoplasm of the cell and is referred to as the soluble methane monooxygenase (sMMO). 3,4 The sMMO is an iron enzyme, and the hydroxylase protein is an ( abg) 2 dimer containing two non-heme binuclear iron clusters that activate the dioxygen and mediate the hydroxyl- Journal of the Chinese Chemical Society, 2004, 51, 1081-1098 1081 Dedicated to Professor Sunney I. Chan on the occasion of his 67 th birthday and his retirement from professional life. * Corresponding author. E-mail: chans@chem.sinica.edu.tw