In Escherichia coli Ammonia Inhibits Cytochrome bo3 But Activates Cytochrome bd-I

Abstract

Interaction of two redox enzymes of Escherichia coli, cytochrome bo3 and cytochrome bd-I, with ammonium sulfate/ammonia at pH 7.0 and 8.3 was studied using high-resolution respirometry and absorption spectroscopy. At pH 7.0, the oxygen reductase activity of none of the enzymes is affected by the ligand. At pH 8.3, cytochrome bo3 is inhibited by the ligand, with 40% maximum inhibition at 100 mM (NH4)2SO4. In contrast, the activity of cytochrome bd-I at pH 8.3 increases with increasing the ligand concentration, the largest increase (140%) is observed at 100 mM (NH4)2SO4. In both cases, the effector molecule is apparently not NH4+ but NH3. The ligand induces changes in absorption spectra of both oxidized cytochromes at pH 8.3. The magnitude of these changes increases as ammonia concentration is increased, yielding apparent dissociation constants Kdapp of 24.3 ± 2.7 mM (NH4)2SO4 (4.9 ± 0.5 mM NH3) for the Soret region in cytochrome bo3, and 35.9 ± 7.1 and 24.6 ± 12.4 mM (NH4)2SO4 (7.2 ± 1.4 and 4.9 ± 2.5 mM NH3) for the Soret and visible regions, respectively, in cytochrome bd-I. Consistently, addition of (NH4)2SO4 to cells of the E. coli mutant containing cytochrome bd-I as the only terminal oxidase at pH 8.3 accelerates the O2 consumption rate, the highest one (140%) being at 27 mM (NH4)2SO4. We discuss possible molecular mechanisms and physiological significance of modulation of the enzymatic activities by ammonia present at high concentration in the intestines, a niche occupied by E. coli.

Bioenergetics and Reactive Nitrogen Species in Bacteria

“…The bd enzymes play other critical roles in microbes [94,[108][109][110][111]. They contribute significantly to the ability of bacteria to resist stresses induced by peroxide [49,[112][113][114][115][116], sulfide [5,[117][118][119][120], ammonia [121], chromate [122], cyanide [117,123]. Due to the fact that the bd oxidases are often found in pathogenic bacteria but absent in humans, they can be used as protein targets for next-generation antimicrobials [43,64,68,[124][125][126][127][128][129][130][131][132][133][134].…”

Membrane-Bound Redox Enzyme Cytochrome bd-I Promotes Carbon Monoxide-Resistant Escherichia coli Growth and Respiration

“…A protective role of cytochrome bd-I against nitric oxide [69][70][71], peroxynitrite [72], and ammonia [73] was reported. Cytochrome bd-I likely provides NO resistance in E. coli for two reasons.…”

“…The reaction likely occurs on the heme d active site, and at least four possible reaction mechanisms have been suggested [76]. Cytochrome bd-I is not only resistant to but also activated by ammonia under alkaline conditions [73]. In this case, ammonia is suggested to react with a few catalytic intermediates of the enzyme.…”

Interaction of Terminal Oxidases with Amphipathic Molecules

“…In contrast to heme–copper oxidases, bd -oxidases do not pump protons, but still generate ∆μH + due to the spatial separation of the reactions proceeding with the consumption and release of protons [ 29 ]. They are highly resistant to the most common inhibitors of heme–copper enzymes [ 30 , 31 ].…”