The Membrane-Associated Methane Monooxygenase (pMMO) and pMMO-NADH:Quinone Oxidoreductase Complex from Methylococcus capsulatus Bath

Author:

Choi Dong-W.12,Kunz Ryan C.1,Boyd Eric S.1,Semrau Jeremy D.3,Antholine William E.4,Han J.-I.3,Zahn James A.2,Boyd Jeffrey M.1,de la Mora Arlene M.5,DiSpirito Alan A.1

Affiliation:

1. Department of Biochemistry, Biophysics, and Molecular Biology

2. Graduate Program in Microbiology

3. Department of Civil and Environmental Engineering, The University of Michigan, Ann Arbor, Michigan 48109-2125

4. Biophysics Research Institute, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

5. Department of Psychology, Iowa State University, Ames, Iowa 50011-3211

Abstract

ABSTRACT Improvements in purification of membrane-associated methane monooxygenase (pMMO) have resulted in preparations of pMMO with activities more representative of physiological rates: i.e., >130 nmol · min −1  · mg of protein −1 . Altered culture and assay conditions, optimization of the detergent/protein ratio, and simplification of the purification procedure were responsible for the higher-activity preparations. Changes in the culture conditions focused on the rate of copper addition. To document the physiological events that occur during copper addition, cultures were initiated in medium with cells expressing soluble methane monooxygenase (sMMO) and then monitored for morphological changes, copper acquisition, fatty acid concentration, and pMMO and sMMO expression as the amended copper concentration was increased from 0 (approximately 0.3 μM) to 95 μM. The results demonstrate that copper not only regulates the metabolic switch between the two methane monooxygenases but also regulates the level of expression of the pMMO and the development of internal membranes. With respect to stabilization of cell-free pMMO activity, the highest cell-free pMMO activity was observed when copper addition exceeded maximal pMMO expression. Optimization of detergent/protein ratios and simplification of the purification procedure also contributed to the higher activity levels in purified pMMO preparations. Finally, the addition of the type 2 NADH:quinone oxidoreductase complex (NADH dehydrogenase [NDH]) from M. capsulatus Bath, along with NADH and duroquinol, to enzyme assays increased the activity of purified preparations. The NDH and NADH were added to maintain a high duroquinol/duroquinone ratio.

Publisher

American Society for Microbiology

Subject

Molecular Biology,Microbiology

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