NARK IS A NITRITE-EXTRUSION SYSTEM INVOLVED IN ANAEROBIC NITRATE RESPIRATION BY ESCHERICHIA-COLI

Escherichia coli can use nitrate as a terminal electron acceptor for anaerobic respiration. A polytopic membrane protein, termed NarK, has been implicated in nitrate uptake and nitrite excretion and is thought to function as a nitrate/nitrite antiporter. The longest-lived radioactive isotope of nitrogen, N-13-nitrate (half-life = 9.96 min) and the nitrite-sensitive fluorophore N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide have now been used to define the function of NarK. At low concentrations of nitrate, NarK mediates the electrogenic excretion of nitrite rather than nitrate/nitrite exchange. This process prevents intracellular accumulation of toxic levels of nitrite and allows further detoxification in the periplasm through the action of nitrite reductase

The caa(3) terminal oxidase of Bacillus stearothermophilus - Transient spectroscopy of electron transfer and ligand binding

The thermophilic bacterium Bacillus stearothermophilus possesses a caa(3)-type terminal oxidase, which was previously purified (De Vrij, W., Heyne, R. I. HL, and Konings, W. N. (1989) Ear. J. Biochem. 178, 763-770). We have carried out extensive kinetic experiments on the purified enzyme by stopped-flow time-resolved optical spectroscopy combined with singular value decomposition analysis. The results indicate a striking similarity of behavior between this enzyme and the electrostatic complex between mammalian cytochrome c and cytochrome c oxidase. CO binding to fully reduced caa(3) occurs with a second order rate constant (k = 7.8 x 10(4) M(-1) S-1) and an activation energy (E* = 6.1 kcal mol(-1)) similar to those reported for beef heart cytochrome c oxidase. Dithionite reduces cytochrome a with bimolecular kinetics, while cytochrome a(3) (and Cu-B) is reduced via intramolecular electron transfer. When the fully reduced enzyme is mixed with O-2, cytochrome a(3), and cytochrome c are rapidly oxidized, whereas cytochrome a remains largely reduced in the first few milliseconds. When cyanide-bound caa(3) is mixed with ascorbate plus TMPD, cytochrome c and cytochrome a are synchronously reduced; the value of the second order rate constant (k = 3 x 10(5) M(-1) S-1 at 30 degrees C) suggests that cytochrome c is the electron entry site. Steady-state experiments indicate that cytochrome a has a redox potential higher than cytochrome c. The data from the reaction with O-2 reveal a remarkable similarity in the kinetic, equilibrium, and optical properties of caa(3) and the electrostatic complex cytochrome c/cytochrome c oxidase