Structural Characterization of the Particulate Methane Monooxygenase

 by Electron Paramagnetic Resonance and X-ray absorption Spectroscopy

 

Chiu-Fou Tseng, 1,2* Huang-Chou Chen, 1,2* Shyue-Chu Ke,3 Steve S.-F. Yu, 1

Sunney I. Chan1,2

 

Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan1

Department of Chemistry, National Tsing-Hua University, Hsin-chu , Taiwan2

Department of Physics, National Dong-Hua University, Hua-lien, Taiwan3

 

The pMMO isolated from Methylococcus capsulatus (Bath) consists of a three-subunit hydroxylase (45, 27 and 23 kDa) and a NADH oxidoreductase (38 kDa).  The hydroxylase is a copper protein, with 15 copper ions arranged in five trinuclear copper clusters.  From the EPR spectrum of the as-isolated copper enriched particulate methane monooxygenase, we observed a type II copper signal with g//=2.24, A//=185G, and g^=2.058 associated with hyperfine splitting of about 150 G centered at g//=2.29.  From 15N labeling experiments, the superhyperfine splittings observed may be assigned to a catalytic center coupled with three nitrogens.

In order to explore the structure of the active site as well as the nature of the reaction intermediate(s) formed at the active site during turnover of the enzyme, we have subjected the pMMO to different levels of reductants such as dithionite, as well as oxidants, including ferricyanide or oxygen under different oxygen tension, and examined the copper ions at various stages of oxidation of the copper clusters.  Both the catalytic and electron transfer clusters (C- and E-clusters, respectively) were examined by EPR spectroscopy and X-ray absorption spectroscopy to distinguish between various multi-oxidation states of the copper clusters.  From the disappearance and the presence of the type II copper signals and superhyperfine splittings with incremental addition of reductive as well as oxidative reagents, we have been able to distinguish between the two catalytic sites from the titration experiments.  Upon fully oxidizing the pMMO by ferricyanide, we observe a g~2.096 isotropic EPR signal, which we have identified and characterized by computer simulation analysis to be associated with a fully oxidized tricopper cluster signal.  With the above results, we have made an important step forward in our efforts toward defining the functional role of some of the copper ions in the catalytic pathway of the pMMO.