Redox Potentiometry Studies of Particulate Methane Monooxygenase: Support for a Trinuclear Copper Cluster Active Site

Abstract

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...

Mutagenesis of the hydrocarbon monooxygenase indicates a metal centre in subunit-C, and not subunit-B, is essential for copper-containing membrane monooxygenase activity

“…Based on the higher resolution Methylocystis strain M structure, an alternate model of this site was proposed involving only PmoC residues (Culpepper & Rosenzweig, 2012;Smith et al, 2011). Alternative models for tricopper and di-iron metal centres have also been proposed by Chan et al (2007) and Martinho et al (2007), respectively. Given the variation between models and the low activity of pMMO protein preparations, the biological relevance of the metal centre(s) in the active pMMO is still contested (reviewed by Culpepper & Rosenzweig, 2012;Semrau et al, 2010).…”

Inactivation of the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath) by acetylene

“…We have proposed that a tricopper cluster located at site D indeed is the catalytic site of pMMO. EPR and other biochemical data have provided strong evidence for the existence of this putative tricopper site, and the involvement of this site in the catalytic mechanism of the enzyme [1,16,38]. A tricopper-peptide complex has recently been prepared based on the PmoA peptide HIHAMLTMGDWD that lines the empty hydrophilic cavity at this site in the protein crystal structure of pMMO from M. capsulatus [16].…”

“…A tricopper-peptide complex has recently been prepared based on the PmoA peptide HIHAMLTMGDWD that lines the empty hydrophilic cavity at this site in the protein crystal structure of pMMO from M. capsulatus [16]. According to model building, the tricopper cluster is sequestered at site D by the ligands in bold in this peptide as well as an additional Glu from PmoA, together with another Glu from PmoC [38]. Upon activation of the Cu I Cu I Cu I -peptide complex by dioxygen in the presence of propene or methane, rapid oxidation of these substrates has been observed at room temperature, with propene converted into propylene oxide and methane into methanol, respectively [16].…”

A Physical Chemist's Expedition to Explore the World of Membrane Proteins

“…In our work, a preparation of the enzyme with the full complement of 15 Cu ions was always used (26). At the active site of this enzyme is a trinuclear copper cluster (24,26,68) that, upon activation by molecular oxygen, mediates the transfer of a singlet oxene to the methane at the active site. A recent crystal structure of pMMO from M. capsulatus did not reveal a trinuclear copper center in the structure (60), but the protein preparation on which the crystal structure was based did not possess enzyme activity.…”

“…Those of us who conduct biophysical measurements are often less circumspect about the quality of the sample than they should be. Fortunately, in the case of pMMO, it was possible to reconcile the biochemical/biophysical data with the crystal structure by introducing the missing metallic cofactors back into the protein scaffold (24,26,68) (Figure 3).…”