The soluble methane monooxygenase and ammonia oxidation in the obligate methanotroph 'Methylosinus trichosporium (OB3b)'
The aim of this project was to isolate and characterise the soluble methane monooxygenase (MMO) from the obligate methanotroph Methylosinus trichosporium (OB3b) and to investigate its role in the oxidation of ammonia. The nature and location of the MMO was shown to be dependent on the availability of copper to the organism. Cells grown in chemostat culture with copper in excess produced a particulate MMO. whereas under conditions of copper stress a soluble MMO is ,produced. This response was independent of the carbon and energy source used for growth (methane or methanol). The soluble MMO was separated into two fractions by DEAE ion exchange chromatography. Each had no MMO activity when assayed individually but had MMO activity when assayed in combination. Fraction A consisted of material that failed to bind to DEAE cellulose and from it component A of the soluble MMOwas purified. Component A had an Mr of 230000 and consisted of three subunits a, B and Y of Mr 54000. 40000 and 18500 respectively. suggesting a a2B2Y2 subunit structure. Component A could replace component A of the soluble MMO of Methylococcus capsulatus (Bath) in assays of pure components of the soluble MMO from this organism and was therefore identified as the hydroxylase component of the enzyme. Fraction C consisted Of material eluted from DEAE cellulose by 0.3 M NaC1, from it component C of the soluble MMO was partially purified. Component C was purified to a point where it consisted predominantly of two proteins of Mr 38000 and 58000. Component C could replace component C of the soluble MMO of Methylococcus capsulatus (Bath) in MMO assays of pure components of the soluble MMO from this organism and was therefore identified as the NADH:acceptor reductase component of the enzyme. The presence of a third component (component B) of the soluble MMO essential for MMO activity was demonstrated. Component B was not purified or isolated from components A or C but it was 'shown to be analogous to component B of the soluble MMO of MethYlococcus capsulatus (Bath). The close functional and physicochemical similarity between the components of the soluble MMOs from Methylosinus trichosporium (OB3b) and Methylococcus capsulatus (Bath) is discussed. as is the distinct difference between the soluble and particulate MMOs from Methylosinus trichosporium (OB3b). The soluble MMOwas shown to oxidise ammonia to hydroxylamine in that: 1. ammonia oxidation required the presence of NAD(P)H for activity as does the soluble MMO; 2. ammonia oxidation was inhibited by acetylene and 8-hydroxyquinoline. specific inhibitors of the soluble MMO. 3. Ammonia oxidation required the presence of both DEAE fractions of the soluble MMO for activity. and U. ammonia oxidation activity was always associated with the soluble MMO and was never present in extracts lacking soluble MMO activity. Hydroxylamine inhibits the soluble MMO (50% at 1,mM) and this was identified as a cause of the cessation of maximum ammonia oxidising activity after 1 minute 1n vitro. Only low levels of hydroxylamine oxidoreductase activity were measured in vitro (> 1 nmol/min/mg) and activity failed to be stimulated by the addition of a number of electron donors.