Respiration and protein synthesis in Escherichia coli membrane-envelope fragments. 3. Electron microscopy and analysis of the cytochromes.

The membranous nature of pellets obtained from broken Escherichia coli spheroplasts by successive centrifugation at 3500 g (P(1)), 20,000 g (P(2)), and 105,000 g (P(3)), has been established by electron microscopy. Spectrophotometric analysis has shown that about 90% of the cytochromes are concentrated in the particulate fractions. The crude ribosomal pellet (P(3)) contained as much of the total cytochromes as did the pellet obtained at 20,000 g (P(2)). The high cytochrome content of P(3) is consistent with its high oxidative activity (1) and the presence of membrane vesicles in this fraction. Analysis at 77 degrees K intensified the optical extinction of all the cytochrome absorption bands, but the degree of intensification was not uniform for each fraction nor for each band within a given fraction. Carbon monoxide had little or no inhibiting effect on NADH oxidation. Reduced plus carbon monoxide difference spectra yielded artifactual absorption bands in the wave length regions where reduced vs. oxidized absorption bands normally occur. Succinate and NADH, either together or separately, reduced nearly all of the cytochromes, indicating that the cytochrome portion of the electron-transport chain is shared by both substrates. A tentative formulation of the electron-transport chain is presented.