The Characterization of the Copper Clusters of the Particulate Methane
Monooxygenase in Methylococcus capsulatus (Bath)
Huang-Chou Chen*,1,2, Steve Sheng-Fa Yu1, Sunney I. Chan1,2
1
Institute of Chemistry, Academia Sinica, Taipei 115, Taiwan
2
Department of Chemistry, National Tsing-Hua University, Hsin-chu 300, Taiwan
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. The functional
form of the enzyme is the fully or partially reduced copper hydroxylase. The pMMO exhibits
unusual substrate specificity: only small normal alkanes are hydroxylated and similar alkenes
epoxidated. In addition, the chemistry is highly regiospecific as well as stereoselective.
Experiments on cryptically chiral ethanes show that the insertion of the active “oxygen” species
occurs with 100% retention of configuration. This result provides the arguments for a concerted
reaction pathway for the hydroxylation, suggesting that the C-H activation might involve a Cu (III)
intermediate in a multi-copper reaction center.
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.