The membrane-associated form of methane mono-oxygenase from Methylococcus capsulatus (Bath) is a copper/iron protein.
A protocol has been developed which permits the purification of a membrane-associated methane-oxidizing complex from Methylococcus capsulatus (Bath). This complex has approximately 5 fold higher specific activity than any purified particulate methane mono-oxygenase (pMMO) previously reported from M. capsulatus (Bath). This efficiently functioning methane-oxidizing complex consists of the pMMO hydroxylase (pMMOH) and an unidentified component we have assigned as a potential pMMO reductase (pMMOR). The complex was isolated by solubilizing intracytoplasmic membrane preparations containing the high yields of active membrane-bound pMMO (pMMO(m)), using the non-ionic detergent dodecyl-beta-D-maltoside, to yield solubilized enzyme (pMMO(s)). Further purification gave rise to an active complex (pMMO(c)) that could be resolved (at low levels) by ion-exchange chromatography into two components, the pMMOH (47, 27 and 24 kDa subunits) and the pMMOR (63 and 8 kDa subunits). The purified complex contains two copper atoms and one non-haem iron atom/mol of enzyme. EPR spectra of preparations grown with (63)Cu indicated that the copper ion interacted with three or four nitrogenic ligands. These EPR data, in conjunction with other experimental results, including the oxidation by ferricyanide, EDTA treatment to remove copper and re-addition of copper to the depleted protein, verified the essential role of copper in enzyme catalysis and indicated the implausibility of copper existing as a trinuclear cluster. The EPR measurements also demonstrated the presence of a tightly bound mononuclear Fe(3+) ion in an octahedral environment that may well be exchange-coupled to another paramagnetic species.
Basu
Piku
P
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, U.K.
Katterle
Bettina
B
Andersson
K Kristoffer
KK
Dalton
Howard
H
D001205:Ascorbic Acid / Q000737:chemistry; D001426:Bacterial Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology*; D003300:Copper / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D007501:Iron / Q000378:metabolism*; D008667:Metalloproteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D020619:Methylococcus capsulatus / Q000201:enzymology*; D009097:Multienzyme Complexes / Q000302:isolation & purification; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D008667:Metalloproteins; D009097:Multienzyme Complexes; D003300:Copper; D007501:Iron; D010105:Oxygenases; C021555:methane monooxygenase; D001205:Ascorbic Acid