Copper ions as inhibitors of protein C of soluble methane monooxygenase of Methylococcus capsulatus (Bath)

Abstract

Copper(I), copper(I1) and silver ions have been shown to be potent inhibitors of purified soluble methane monooxygenase (MMO) of Methylococcus cupsulatus (Bath). A weaker inhibition has been observed with zinc and cadmium ions. Proteins A and B of soluble MMO are unaffected by copper but protein C is rapidly and irreversibly inhibited. The site of copper inhibition has been shown to be primarily at the iron-sulphur centre of protein C with a secondary effect at the FAD centre when the copper(1I):protein C ratio is high. Copper appears to bring about the inhibition of soluble MMO by interacting with protein C to disrupt the protein structure causing, firstly, the loss of the iron-sulphur centre, preventing the transfer of electrons from protein C to protein A, and secondly, the loss of FAD preventing the protein from accepting electrons from NADH. Inhibition and spectral data are provided to support this thesis. The inactivation of protein C is associated with the tight binding of four Cu atoms to each protein C molecule.These data extend our knowledge of how copper, which is known to have a key role in the cellular location of MMO, interacts with and rapidly and irreversibly inactivates the soluble form of this enzyme.The cellular location of the enzyme methane monooxygenase (MMO) of the obligate methanotroph Methylococcus cupsulatus (Bath), which catalyses the oxidation of methane to methanol, (Eqn 1 [l, 21) has been shown to be dependent on the amount of copper available to the cells during growth.It has recently been demonstrated that at low copper: biomass ratios the predominant form of the enzyme occurs in the soluble fraction of cell extracts and that at high copper: biomass ratios the soluble MMO is inactivated and the particulate, membrane-bound MMO becomes activated [2]. Copper has been shown to markedly increase the activity of particulate MMO when NADH is supplied as electron donor [2, 31 and this effect has also been observed for particulate extracts of 'Methylosinus' sp. strain . Loss of soluble MMO activity on switching to high copper (1.2 mg Cu2' 1 -I ) medium occurs very rapidly and may be due to inhibition of the enzyme by copper [2].The two forms of MMO show marked differences with regard to substrate specificity: the soluble enzyme will oxidize a wide range of alkanes, alkenes, alicyclics and aromatic compounds [5] whereas the particulate enzyme will only oxidize alkanes up to pentane and does not oxidize aromatics under the conditions used in these experiments [7, 81, the predominant form of the enzyme would have been particulate MMO, which is readily inhibited by metal-binding compounds [I]. There have been no reports of inhibition of the soluble MMO by metal ions but it has been demonstrated that soluble MMO is not sensitive to metal chelators [I].Soluble MMO is a multiprotein enzyme consisting of three proteins, all of which are essential for activity [9]. All three proteins have been purified to near homogeneity [lo-141. Protein A is a non-haem-iron protein of M , 210000, which interacts ...

Alkene monooxygenase from Mycobacterium: a multicomponent enzyme

“…The observed inhibition pattern for A M 0 of Mycobacterium E3 is very similar to the inhibition patterns reported for the soluble methane monooxygenases of Methylococcus capsulatus (Bath) and Methylosinus trichosporiurn OB3b (Scott et al, 198 1 revealed the presence of an unusual non-haem iron centre (Green & Dalton, 1988). The iron in component A could be removed by dialysis against 8-hydroxyquinoline, resulting in an inactive protein.…”

Two‐Component Flavin‐dependent Pyrrole‐2‐carboxylate Monooxygenase from Rhodococcus Sp.

“…Thus, it seems to resemble the small protein B of the multicomponent soluble methane monooxygenase from methanotrophic bacteria. In the case of Methylococcus capsulatus (Bath) this 16-kDa protein is devoid of prosthetic groups and converts methane monooxygenase from an oxidase to an oxygenase (Green and Dalton, 1985;Lipscomb, 1994). However, no sequence similarity was found between protein B and the small subunits of the two-component monooxygenases (Pilkington et al, 1990;Cardy et al, 1991 ;Prieto and Garcia, 1994;Takizawa et al, 1995;this study).…”

Sequence‐alignment modelling and molecular docking studies of the epoxygenase component of alkene monooxygenase from Nocardia corallina B‐276

“…A-naphthylamine in 30% (by vol.) acecycles [7]. After the fourth cycle the iron content was reduced tic acid.…”

“…A DEAE-cellubelieve particular residues are able to account for the difference lose (Whatman) FPLC column (250 mmϫ56 mm, 120 ml) in stereoselectivity observed between AMO [6] and soluble equilibrated with buffer B was used for initial seperation of the MMO [7].…”

“…The prosthetic groups were integrated by transferring the coordinates of groups from the template structure after samples. 10 µM AMO epoxygenase in buffer B was depleted of iron by treatment of protein to four freeze-thaw cycles [7]. The superimposition of the model on to the template [13].…”