Subunit sites of oxidative inactivation of Escherichia coli F1-ATPase by HOCl.

Cellular inactivation of Escherichia coli by the neutrophil-generated toxin, hypochlorous acid, is accompanied by inactivation of its plasma membrane-localized F1-ATPase. The nature of oxidative damage leading to inactivation of this enzyme was probed by SDS-PAGE and 2D-gel electrophoresis and by hybrid reconstitution studies using purified subunits from untreated and extensively oxidized bacteria. The data indicate that inactivation is due to selective oxidation of a few highly vulnerable sites; although damage occurred to each of the alpha, beta, and gamma-subunits required for soluble ATP hydrolase activity, the extent of damage was insufficient to alter their electrophoretic properties.

Hypochlorous acid and myeloperoxidase-catalyzed oxidation of iron-sulfur clusters in bacterial respiratory dehydrogenases.

Hypochlorous acid and related oxidants derived from myeloperoxidase-catalyzed reactions contribute to the microbicidal activities of phagocytosing neutrophils and monocytes. Microbial iron-sulfur (Fe/S) clusters have been suggested as general targets of myeloperoxidase-derived oxidations, but no susceptible Fe/S site has yet been identified. In this study, the effects of HOCl and myeloperoxidase-catalyzed peroxidation of chloride ion upon EPR-detectable Fe/S clusters in Escherichia coli and Pseudomonas aeruginosa were examined. Increasing amounts of oxidant produced progressive loss of signal amplitudes from the S-1 and S-3 Fe/S clusters of succinate:ubiquinone oxidoreductase in respiring membrane fragments. These changes were compared to loss of microbial viability, succinate uptake rates, succinate dehydrogenase activity and succinate-dependent respiration. The amounts of oxidant required to destroy Fe/S clusters exceeded the amounts required to kill organisms or inhibit respiratory function by factors of four or five. Power saturation characteristics of the S-1 signal indicated that the S-2 signal was also resistant to modification, even in highly oxidized membranes. Loss of succinate-dependent respiration was closely associated with HOCl and myeloperoxidase-mediated microbicidal activity against P. aeruginosa and was also an early event in the oxidant-mediated metabolic dysfunctions of E. coli. However, these effects were not caused by the destruction of the Fe/S clusters within the succinate:ubiquinone oxidoreductase. Rather, the major respiration-inhibiting lesion(s) appeared to reside at points in the respiratory chain between the Fe/S clusters and the ubiquinone reductase site.

General mechanism for the bacterial toxicity of hypochlorous acid: abolition of ATP production.

The adenylate energy charges (EC) of Escherichia coli 25922, Pseudomonas aeruginosa 27853, and Streptococcus lactis 7962 rapidly fell in nutrient-rich media from values in excess of 0.9 to below 0.1 when the organisms were exposed to lethal levels of HOCl. The same cells maintained in energy-depleted states were incapable of attaining normal EC values necessary for biosynthesis and growth when challenged with nutrient energy sources after HOCl exposure. These changes correlated quantitatively with loss of replicative capabilities. Initial rates of transport of glucose, succinate, and various amino acids that act as respiratory substrates and the ATP hydrolase activity of the F1 complex from the ATP synthase of E. coli 25922 also declined in parallel with or preceded loss of viability. These results establish that cellular death is accompanied by complete disruption of bacterial ATP production by both oxidative and fermentative pathways as a consequence of inhibition of inner membrane bound systems responsible for these processes.

Leukocytic oxygen activation and microbicidal oxidative toxins.

Following a brief introduction of cellular response to stimulation comprising leukocyte activation, three major areas are discussed: (1) the neutrophil oxidase; (2) myeloperoxidase (MPO)-dependent oxidative microbicidal reactions; and (3) MPO-independent oxidative reactions. Topics included in section (A) are current views on the activation mechanism, redox composition, structural and topographic organization of the oxidase, and its respiratory products. In section (B), emphasis is placed on recent research on cidal mechanisms of HOCl, including the oxidative biochemistry of active chlorine compounds, identification of sites of lesions in bacteria, and attendant metabolic consequences. In section (C), we review the (bio)chemistry of H2O2 and .OH microbicidal reactions, with particular attention being given to addressing the controversial issue of probe methods to identify .OH radical and critical assessment of the recent proposal that MPO-independent killing arises from site-specific metal-catalyzed Fenton-type chemistry.

Viability and metabolic capability are maintained by Escherichia coli, Pseudomonas aeruginosa, and Streptococcus lactis at very low adenylate energy charge.

Metabolic regulation by nucleotides has been examined in several bacteria within the context of the adenylate energy charge (EC) concept. The ECs of bacteria capable of only fermentative metabolism (Streptococcus lactis and the ATPase-less mutant Escherichia coli AN718) fell to less than 0.2 under carbon-limiting conditions, but the bacteria were able to step up the EC to greater than 0.8 upon exposure to nutrient sugars. Similarly, nongrowing E. coli 25922, whose EC had been artificially lowered to less than 0.1 by the addition of the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), was able to immediately step up the EC to 0.8 to 0.9 upon the addition of glucose but was unable to respond to respiratory substrates. The EC of respiring bacteria (E. coli 25922 and Pseudomonas aeruginosa 27853) fell to 0.3 to 0.4 under certain limiting growth conditions, but the bacteria also responded immediately when challenged with succinate to give EC values greater than 0.8. These bacteria could not step up the EC with respiratory substrates in the presence of CCCP. For all bacteria, the loss of the ability to step up the EC was attributable to the loss of nutrient transport function. Mixtures of viable and HOCl-killed E. coli 25922 were able to step up the EC in proportion to the fraction of surviving cells. The data indicate that nucleotide phosphorylation levels are not regulatory in nongrowing bacteria but that the EC step-up achievable upon nutrient addition may be an accurate index of viability.

Hypochlorous acid-promoted loss of metabolic energy in Escherichia coli.

Oxidation of Escherichia coli by hypochlorous acid (HOCl) or chloramine (NH2Cl) gives rise to massive hydrolysis of cytosolic nucleotide phosphoanhydride bonds, although no immediate change occurs in either the nucleotide pool size or the concentrations of extracellular end products of AMP catabolism. Titrimetric curves of the extent of hydrolysis coincide with curves for loss of cell viability, e.g., reduction in the adenylate energy charge from 0.8 to 0.1-0.2 accompanies loss of 99% of the bacterial CFU. The oxidative damage caused by HOCl is irreversible within 100 ms of exposure of the organism, although nucleotide phosphate bond hydrolysis requires several minutes to reach completion. Neither HOCl nor NH2Cl reacts directly with nucleotides to hydrolyze phosphoanhydride bonds. Loss of viability is also accompanied by inhibition of induction of beta-galactosidase. The proton motive force, determined from the distribution of 14C-radiolabeled lipophilic ions, declines with incremental addition of HOCl after loss of respiratory function; severalfold more oxidant is required for the dissipation of the proton motive force than for loss of viability. These observations establish a causal link between loss of metabolic energy and cellular death and indicate that the mechanisms of oxidant-induced nucleotide phosphate bond hydrolysis are indirect and that they probably involve damage to the energy-transducing and transport proteins located in the bacterial plasma membrane.