The organization and properties of the cytochromes of the respiratory chain of Escherichia coli
Creators: name: Pudek, Morris Romuald
Publisher: name: University of British Columbia
Year: 2010

description: Escherichia coli contains cytochromes a₁, b₅₅₆, b₅₅₈, d, and o with smaller amounts of a c-type cytochrome(s) and cytochrome b₅₆₂ in the membrane bound electron transport system. The levels of these components vary with the growth, conditions. Cytochromes d and o function as terminal oxidases. Membrane particles prepared from cells grown to the stationary phase contain higher levels of cytochrome d and lower levels of cytochrome o than cells grown to the exponential phase. The NADH oxidase activity of particles which contain the higher levels of cytochrome d is less sensitive to inhibition by cyanide. The second order rate constant for the formation of cyanocyto-chrome d in particles oxidizing NADH is found to correlate with the rate constant determined for the inhibition of NADH oxidase activity by cyanide. The magnitude of the second order rate constant for the formation of cyanocytochromesd is directly proportional to the rate of electron flux through cytochrome d. The oxidized form of cytochrome d with an alpha absorption peak at 648 nm and the reduced form with an alpha absorption peak at 628 nm are not direct oxidation-reduction products of each other. Cytochrome d goes through an intermediate form (d*) in the normal oxidation-reduction cycle. This form, which has no apparent alpha absorption peak, is the species which reacts with cyanide. Cytochrome d can be trapped as this intermediate at subzero temperatures in particles oxidizing ascorbate in the presence of phenazine methosulfate. The nature of this intermediate is discussed in relation to the cytochrome oxidase reaction mechanism. The greater sensitivity of NADH oxidase activity to inhibition by cyanide in cells containing low levels of cytochrome d is due to the higher steady state level of the intermediate species reactive with cyanide caused by the greater rate of electron flux through the smaller cytochrome d pool in these cells. The contribution of cytochrome d and cytochrome o to the NADH and succinate oxidase activity appears to be directly proportional to their respective concentrations in the membrane bound respiratory system. The midpoint oxidation-reductions potentials of the cytochromes of the respiratory chain of Escherichia coli were determined. Cytochromes d and&gt;a₁ have midpoint oxidation-reduction potentials of +260 mV and +147 mV, respectively. Cytochrome b can be resolved into two major components by difference spectroscopy at 77°K and by potentiometric titrations. Cytochrome b₅₅₈ and cytochrome b₅₅₆ have midpoint oxidation-reduction potentials of +165 mV and +35 mV, respectively, in membranes from exponential phase cells. The potentials of the cytochrome b components may vary with the growth phase. Cytochrome b₅₅₈ increases in amount relative to cytochrome b₅₅₆ in the transition from the exponential to the stationary phase of growth. Partial compartmentalizatiori of the NADU, and succinate oxidase systems is indicated by the kinetics of reduction of the b cytochromes and by the greater inhibition by 2-heptyl-4 hydroxyquinoline N-oxide of NADH oxidase compared with succinate oxidase. This inhibitor appears to block electron trans port before and after both cytochromes b₅₅₆ and b₅₅₈. A scheme for the arrangement of the cytochromes in the NADH and succinate oxidase pathways in respiratory particles of E. coli is presented.