The succinate dehydrogenase ofEscherichia coli: resolution as a major membrane-bound immunogen possessing covalently bound flavin

Succinate dehydrogenase (EC 1.3.99.1) is a key component of both eukaryotic and prokaryotic electron transport systems. This membrane-bound flavoprotein catalyses the oxidation of succinate to fumarate and transfers reducing equivalents to the respiratory chain. The succinate dehydrogenase from both mammalian and yeast mitochondria [1,2] and from the photosynthetic bacterium Rhodospirillum rubrum [3,4] have been well characterized. The properties of similar enzymes from two Gram-positive bacteria, Bacillus subtilis [5] and Micrococcus lysodeikticus [6] have also been described. However, the succinate dehydrogenase of the model Gram-negative bacterium Escherichia coli has proved more refractory to detailed analysis [7,81. A relatively new and productive approach to the study of bacterial membranes and their constituents is that based upon the high-resolution two-dimensional technique of crossed immunoelectrophoresis (CIE). Recently, a complex CIE reference profile was established for membranes of E. coli ML308-225. In the course of these studies, many of the 50 or so resolved antigens were characterized but no antigen displaying succinate dehydrogenase activity was detected, despite its earlier resolution in other systems (reviewed in [9]). We have pursued the immunochemical approach to the study of bacterial succinate dehydrogenase, and in this paper report on the CIE analysis of membranes derived from cells of E. coli ML308-225 grown aerobically in the presence of the ( rad io labe led) F A D / F M N precurso r [14C]riboflavin. We show that two previously unidentified antigens (Nos. 22 and 23) of the CIE reference profile [10,11] possess covalently bound flavin a feature characteristic of succinate dehydrogenase isolated from other sources [1], and that both express succinate dehydrogenase activity under appropriate conditions. Some of the molecular properties of these two flavoenzymes are documented in a companion paper [12]. A preliminary account of this work has been presented [13].