Abstract
AbstractThe enzyme methane monooxygenase (MMO), generated in methanotrophs, catalyzes the conversion of methane to methanol for use as the energy and carbon source. Two distinct forms of MMO are known to exist at different cellular locations, a cytoplasmic (soluble) MMO and a membrane-bound (particulate) MMO. The sMMO has been purified from several methanotrophs and has been characterized biochemically and genetically. The pMMO is less well studied than the sMMO because of the instability of the pMMO upon removal of the polypeptides from the membrane lipids. The pMMO contains at least three subunits, of approximately 45, 27, and 23 kDa in molecular mass. In this work we identified these protein subunits with the modern proteomic tool of one-dimensional SDS-PAGE and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The three separated pMMO subunits of 45, 27, and 23 kDa were observed on the SDS-PAGE and identified as the expression gene products of the pmoB, pmoC, and pmoA, respectively. In the gel of the membrane proteins, other bands were also analyzed. The band of 38 kDa, which has been thought to be a post-translation modification product of the pmoB, appeared as a major band on the gel and identified as a PmoB fragment. A protein fragment was also observed at molecular weight 29.7 kDa, which was identified and characterized as CbbQ. The remaining the fragments appearing at low molecular weights were also identified as the PmoB fragment arising from the protease degradation.We observed N-terminal acetylation of PmoC and PmoA by tandem mass spectrometry. The Edman degradation method was performed on the N-terminal sequences of these pMMO subunits in order to compare with the mass spectrometric results. The subunit of 45 kDa contained a sequence with HGEKS from the N terminus. Unfortunately, the other two subunits could not be directly determined by Edman degradation method due to the properties of the acetylation of these subunits.By considering the outer membrane fraction as well as transmembrane domain, the in-gel tryptic digestion of membrane proteins could only be observed for the peptides released from the exposed domains of the membrane (more hydrophilic portions) by mass spectrometry. Due to multiple cleavage sites and smaller cleavage fragments, the chymotrypsin was chosen for in-gel digestion to cover the peptides in the membrane portion.According to these direct information regarding cytoplasmic or transmembrane structure, we conclude that only peptides in the outer soluble portion from PmoB were observed. None of the tryptic digested peptides from the membrane bound PmoC and PmoA was observed. We have used this approach to elucidate the topological structure of the pMMO in the membrane.This study reports the application of a method that combines modern electrophoresis with mass spectrometry to the pMMO problem may be the first case in protein level. This might well be the first application of this methodology to a membrane protein.