Antibiotics induce redox-related physiological alterations as part of their lethality.

Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal. Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part by inducing production of damaging reactive species. This notion has been supported by many groups but has been challenged recently. Here we robustly test the hypothesis using biochemical, enzymatic, and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular H2O2 sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species to demonstrate that antibiotics broadly induce redox stress. Subsequent gene-expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supraphysiological levels of H2O2. We next developed a method to quantify cellular respiration dynamically and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that overexpression of catalase or DNA mismatch repair enzyme, MutS, and antioxidant pretreatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing. Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions but could be enhanced by exposure to molecular oxygen or by the addition of alternative electron acceptors, indicating that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that, downstream of their target-specific interactions, bactericidal antibiotics induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality.

Autocatalytic O2 cleavage by an OCO3 trianionic pincer Cr(III) complex: isolation and characterization of the autocatalytic intermediate [Cr(IV)]2(µ-O) dimer.

Synthetic and kinetic experiments designed to probe the mechanism of O(2) activation by the trianionic pincer chromium(III) complex [(t)BuOCO]Cr(III)(THF)(3) (1) (where (t)BuOCO = [2,6-((t)BuC(6)H(3)O)(2)C(6)H(3)](3-), THF = tetrahydrofuran) are described. Whereas analogous porphyrin and corrole oxidation catalysts can become inactive toward O(2) activation upon dimerization (forming a µ-oxo species) or product inhibition, complex 1 becomes more active toward O(2) activation when dimerized. The product from O(2) activation, [(t)BuOCO]Cr(V)(O)(THF) (2), catalyzes the oxidation of 1 via formation of the µ-O dimer {[(t)BuOCO]Cr(IV)(THF)}(2)(µ-O) (3). Complex 3 exists in equilibrium with 1 and 2 and thus could not be isolated in pure form. However, single crystals of 3 and 1 co-deposit, and the molecular stucture of 3 was determined using single-crystal X-ray crystallography methods. Variable (9.5, 35, and 240 GHz) frequency electron paramagnetic resonance spectroscopy supports the assignment of complex 3 as a Cr(IV)-O-Cr(IV) dimer, with a high (S = 2) spin ground state, based on detailed computer simulations. Complex 3 is the first conclusively assigned example of a complex containing a Cr(IV) dimer; its spin Hamiltonian parameters are g(iso) = 1.976, D = 2400 G, and E = 750 G. The reaction of 1 with O(2) was monitored by UV-visible spectrophotometry, and the kinetic orders of the reagents were determined. The reaction does not exhibit first-order behavior with respect to the concentrations of complex 1 and O(2). Altering the THF concentration reveals an inverse order behavior in THF. A proposed autocatalytic mechanism, with 3 as the key intermediate, was employed in numerical simulations of concentration versus time decay plots, and the individual rate constants were calculated. The simulations agree well with the experimental observations. The acceleration is not unique to 2; for example, the presence of OPPh(3) accelerates O(2) activation by forming the five-coordinate complex trans-[(t)BuOCO]Cr(III)(OPPh(3))(2) (4).

Catalytic aerobic oxidation by a trianionic pincer Cr(III)/Cr(V) couple.

Aerobic oxidation that incorporates both O atoms into a substrate (PPh(3)) is achieved by employing a Cr(III)/Cr(V)[triple bond]O catalytic couple. A terphenyl trianionic pincer ligand stabilizes a high oxidation state Cr(V)[triple bond]O complex, and both the reduced (Cr(III), IR/X-ray) and oxidized (Cr(V)[triple bond]O, electron paramagnetic resonance/IR/X-ray) participants in the catalytic cycle are characterized.