Chemical cross-linking study of complex formation between methylamine dehydrogenase and amicyanin from Paracoccus denitrificans.
Two soluble periplasmic redox proteins from Paracoccus denitrificans, the quinoprotein methylamine dehydrogenase and the copper protein amicyanin, form a weakly associated complex that is critical to their physiological function in electron transport [Gray, K. A., Davidson, V. L., & Knaff, D. B. (1988) J. Biol. Chem. 263, 13987-13990]. The specific interactions between methylamine dehydrogenase and amicyanin have been studied by using the water-soluble cross-linking agent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). Treatment of methylamine dehydrogenase alone with EDC caused no intermolecular cross-linking but did cause intramolecular cross-linking of this alpha 2 beta 2 oligomeric enzyme. The primary product that was formed contained one large and one small subunit. Methylamine dehydrogenase and amicyanin were covalently cross-linked in the presence of EDC to form at least two distinct species, which were identified by nondenaturing polyacrylamide gel electrophoresis (PAGE). The formation of these cross-linked species was dependent on ionic strength, and the ionic strength dependence was much greater at pH 6.5 than at pH 7.5. The effects of pH and ionic strength were different for the different cross-linked products. SDS-PAGE and Western blot analysis of these cross-linked species indicated that the primary site of interaction for amicyanin was the large subunit of methylamine dehydrogenase and that this association could be stabilized by hydrophobic interactions. In light of these results a scheme is proposed for the interaction of amicyanin with methylamine dehydrogenase that is consistent with previous data on the physical, kinetic, and redox properties of this complex.
Kumar
M A
MA
Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216-4505.
Davidson
V L
VL
D001400:Azurin / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005022:Ethyldimethylaminopropyl Carbodiimide; D008970:Molecular Weight; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000378:metabolism*; D010231:Paracoccus denitrificans / Q000201:enzymology*; D011485:Protein Binding
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; C017079:mauC protein, Methylobacterium extorquens; D001400:Azurin; C021567:methylamine dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors; D005022:Ethyldimethylaminopropyl Carbodiimide