A Re-examination of the Cytochromes of Escherichia coli using Fourth-order Finite Difference Analysis: Their Characterization under Different Growth Conditions and Accumulation during the Cell Cycle

The cytochromes of Escherichia coli K12 have been studied using low-temperature (77 K) difference spectrophotometry. Numerical (i.e. fourth-order finite difference) analysis resolved the a band of reduced minus oxidized spectra of whole cells from aerobically grown cultures into five components, with absorption maxima at 548, 5 5 1.5, 5 5 5. 5, 560 and 563 nm. Using the same differencing intervals, numerical analysis of cells grown under oxygen-limited conditions revealed only two components, at 555.5 and 559 nm. Similar analysis of cells grown anaerobically with fumarate as electron acceptor revealed four absorption maxima, at 548, 550-5, 5 5 5- 5 and 559 nm. Membrane particles from aerobically grown cells showed the same absorption bands as intact cells; preliminary evidence was obtained for a component of the 555.5 nm band that could be relatively easily washed from the membrane. The contribution of cytochrome o or its CO-liganded form to the a region could not be determined by numerical analysis. We conclude, in contrast to a previous application of numerical analysis to the cytochromes of E. coli, that growth under anaerobic or oxygen-limited conditions results in the appearance of cytochromes spectrally distinct from those in aerobically grown cells. An attempt has been made to reconcile the presence of multiple components detected in the a region by numerical analysis of aerobically grown cells with the diverse components described by others. Quantification of cytochromes revealed by numerical analysis in aerobically grown cells separated into size (and thus age) classes by zonal centrifugation showed that the major components accumulated continuously, probably exponentially, throughout the cell cycle