Particulate methane monooxygenase (pMMO) is a complex membrane protein (abg) that-in our hands (see below)-contains approximately 15 copper ions, [1,2] sequestered into catalytic trinuclear copper clusters (C clusters) and electrontransfer copper ions known as E clusters. [3,4] We have proposed that the C clusters mediate alkane hydroxylation and dioxygen reduction chemistry, while the E clusters provide a buffer of reducing equivalents to rapidly rereduce the C cluster copper ions during turnover. Herein, we provide additional support for our hypothesis of a trinuclear copper cluster as the hydroxylation site of pMMO.[4]The EPR spectrum for the as-isolated pMMO in pMMOenriched membranes typically consists of a superposition of signals: a classical type 2 Cu II signal at g av % 2.12, with Cu hyperfine structure in the parallel region (g k = 2.24) and 14 N superhyperfine structure in the perpendicular region (g ? = 2.059), and an almost featureless isotropic signal centered at g % 2.1 that we have assigned to a trinuclear Cu II Cu II Cu II cluster. [4,5] Unlike the type 2 Cu II signal, the signal of the cluster does not saturate at high microwave power.[5]The number and type of metals in pMMO remains an area of intense controversy. [4,[6][7][8][9][10] Others have implicated one or two iron atoms, [6][7][8][9][10] while the X-ray crystal structure of pMMO from Methylococcus capsulatus (Bath) reveals a (abg) 3 trimer, with only one mononuclear copper center, one dinuclear copper cluster, and one zinc ion per abg monomer. [12] No trinuclear copper cluster is observed. We have speculated, however, that as the enzyme preparation on which the X-ray crystal structure is based does not display activity, it may lack the full complement of copper ions to be functional.In principle, the debate over the existence of a trinuclear copper cluster could be settled by redox potentiometry. The copper sites in pMMO would be predicted to have distinct redox potentials, and thus by reducing the protein at different cell potentials the spectroscopic features for each site could be separated and individually assigned. With this goal in mind, EPR data for pMMO-enriched membranes (80-90 % pMMO) have been obtained after equilibration in an electrochemical cell at fixed potentials.Potentiometric titrations of the copper centers were performed by poising the electrochemical cell at + 120 mV and adding small amounts of sodium dithionite to reduce the enzyme gradually in the presence of mediators. The E cluster copper ions remained reduced, as manifested by the appearance of only the signals for the C clusters in the EPR spectrum. The redox potential(s) of the E cluster copper ions are significantly higher than + 120 mV. Reduction of the copper ions of the C clusters occurred at much lower potentials. The type 2 Cu II EPR signal decreased gradually in intensity with increasingly more negative potentials beginning at + 18.3 mV. At a cell potential of À121.3 mV, the spectrum eventually gave way to an isotropic signal centered at g = 2.05 (Figure...
