THE PARTICULATE METHANE MONOOXYGENASE FROM METHYLOCOCCUS-CAPSULATUS (BATH) IS A NOVEL COPPER-CONTAINING 3-SUBUNIT ENZYME - ISOLATION AND CHARACTERIZATION

The particulate methane monooxygenase (pMMO) is known to be very diffi cult to study mainly due to its unusual activity instability in vitro, By cultivating Methylococcus capsulatus (Bath) under methane stress c onditions and high copper levels in the growth medium, membranes highl y enriched in the pMMO with exceptionally stable activity can be isola ted from these cells, Purified and active pMMO can be subsequently obt ained from these membrane preparations using protocols in which an exc ess of reductants and anaerobic conditions were maintained during memb rane solubilization by dodecyl beta-D-maltoside and purification by ch romatography, The pMMO was found to be the major constituent in these membranes, constituting 60-80% of total membrane proteins, The dominan t species of the pMMO was found to consist of three subunits, alpha,be ta and gamma, with an apparent molecular mass of 45, 26, and 23 kDa, r espectively, A second species of the PMMO, a proteolytically processed version of the enzyme, was found to be composed of three subunits, al pha', beta, and gamma, with an apparent molecular mass of 35, 26, and 23 kDa, respectively, The alpha and alpha' subunits from these two for ms of the pMMO contain identical N-terminal sequences, The gamma subun it, however, exhibits variation in its N-terminal sequence, The pMMO i s a copper-containing protein only and shows a requirement for Cu(I) i ons, Approximately 12-15 Cu ions per 94-kDa monomeric unit were observ ed, The pMMO is sensitive to dioxygen tension, On the basis of dioxyge n sensitivity, three kinetically distinct forms of the enzyme can be d istinguished, A slow but air-stable form, which is converted into a '' pulsed'' state upon direct exposure to atmospheric oxygen pressure, is considered as type I pMMO, This form was the subject of our pMMO isol ation effort, Other forms (types II and III) are deactivated to variou s extents upon exposure to atmospheric dioxygen pressure, Under inacti vating conditions, these unstable forms release protons to the buffer (similar to 10 H+/94-kDa monomeric unit) and eventually become complet ely inactive.