Abstract
The work described in this thesis is directed study toward understanding the structure and function of the pMMO from Methylococcus capsulatus (Bath). A variety of modern techniques have been brought to bear on this problem. An important development has been the development of a hollow-fiber bioreactor and fermentation technology toward scaling up the growth of the methanotropic bacteria. This new technology has allowed the purification of highly active pMMO in membranes, as well as in-depth biochemical/biophysical characterization of the membrane protein after the purification of the highly active pMMO. In order to explore the structure of the active site as well as the nature of the reaction intermediate(s) formed at the active site during turnover of the enzyme, we have subjected the pMMO to different levels of reductants, oxidants and suicide substrate acetylene under dioxygen tensions and looked for change at various stages of oxidation of the copper clusters. Both the catalytic and electron transfer clusters (C- and E-clusters, respectively) have been examined by EPR spectroscopy and X-ray K-edge absorption to distinguish between various multi-oxidation states of the copper clusters. To date, EPR and X-ray absorption measurements have confirmed the classification of the 15 copper ions into 3 trinuclear copper clusters for electron transfer (E-clusters) and 2 trinuclear copper clusters for dioxygen chemistry and alkane hydroxylation (C-clusters). Further purification by membrane solubilization in dodecyl b-D maltoside followed by fractionation of the protein-detergent complexes according to molecular size using gel filtration chromatography yielded the pMMO-detergent complex in good yield and high homogeneity. The purified pMMO-detergent complex has not only been identified by mass finger printing, but also by the determination of the actual molecular mass (99kDa) of the pMMO by MALDI-TOF mass analysis. Finally, ATR-FTIR spectroscopy combined with limited proteolysis has provided the direct evidence for the presence of a-helices in the membrane-embedded domains of pMMO.