Prior, Stephen David (1985) The effect of copper ions on methane oxidation by the obligate methylotroph 'Methylococcus capsulatus' (Bath). PhD thesis, University of Warwick.

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Abstract

The work presented in this thesis provided the first report of membrane- bound methane monooxygenase (MMO) activity in the obligate methylotroph Methylococcus capsulatus (Bath). Experiments in batch and continuous culture showed that the intracellular location of the MMO enzyme was regulated by the copper:biomass ratio. Membrane-bound (particulate) MMO activity was associated with a high copper:biomass ratio, whereas, at low copper:biomass ratios the soluble form of the enzyme predominates. Growth of the organism on methanol showed that the expression of the particulate form of the enzyme was constitutive and that expression of the enzyme increased with increasing concentrations of copper in the growth medium.

Thin-section electron microscopy of M. capsulatus (Bath) grown on methane and methanol showed that the cell exhibited changes in intracellular morphology which could be correlated with the presence or absence of particulate MMO activity, although a direct connection between particulate MMO activity and intracytoplasmic membranes was not demonstrated. The change in intracellular location of the MMO was accompanied by changes in the protein banding pattern on SDS- polyacrylamide gels. Particulate MMO activity was characterized by an increase in at least 3 major proteins in the membrane fractions of cell extracts.

Addition of copper ions to chemostat cultures of Methylococcus capsulatus expressing wholly soluble MMO activity led to a rapid "switch-off" of soluble MMO activity and an equally rapid increase in particulate MMO activity. Studies on in vitro MMO activity demonstrated that particulate MMO activity was stimulated by the addition of copper ions to the assay system; however, addition of copper ions to fractions expressing soluble MMO activity led to a rapid inhibition of this form of the enzyme by irreversible inactivation of the NADH acceptor:reductase activity of the protein complex. The stimulation of particulate MMO activity by copper ions was found to be pH dependent due to the binding of the copper ions to the sodium/potassium phosphate buffer used in the assay system.

The particulate MMO was compared with the soluble enzyme from M. capsulatus (Bath) and was shown to differ in several respects including: inhibitor sensitivity, substrate specificity and activity in the presence of various metal ions. Studies using radiolabelled acetylene demonstrated that this compound appeared to act as a suicide-substrate for both forms of the enzyme by irreversibly binding to the active site of the proteins, a method for the partial purification of the particulate enzyme was developed which provided some stabilization of the protein. Studies on copper uptake by the organism demonstrated that this process was energy-dependent and occurred very rapidly when copper was added to copper-limited cultures of the organism.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QD Chemistry Q Science > QR Microbiology
Library of Congress Subject Headings (LCSH):	Methanotrophs -- Research, Gram-negative bacteria, Copper ions
Official Date:	October 1985
Dates:	Date Event October 1985 Submitted
Institution:	University of Warwick
Theses Department:	Department of Biological Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Dalton, Howard
Sponsors:	Science and Engineering Research Council (Great Britain)
Extent:	xiii, 212 leaves : illustrations
Language:	eng

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