Chlorite dismutases (Clds) convert chlorite to O2 and Cl–, stabilizing heme in the presence of strong oxidants and forming the O=O bond with high efficiency. The enzyme from the pathogen Klebsiella pneumoniae (KpCld) represents a subfamily of Clds that share most of their active site structure with efficient O2-producing Clds, even though they have a truncated monomeric structure, exist as a dimer rather than a pentamer, and come from Gram-negative bacteria without a known need to degrade chlorite. We hypothesized that KpCld, like others in its subfamily, should be able to make O2 and may serve an in vivo antioxidant function. Here, it is demonstrated that it degrades chlorite with limited turnovers relative to the respiratory Clds, in part because of the loss of hypochlorous acid from the active site and destruction of the heme. The observation of hypochlorous acid, the expected leaving group accompanying transfer of an oxygen atom to the ferric heme, is consistent with the more open, solvent-exposed heme environment predicted by spectroscopic measurements and inferred from the crystal structures of related proteins. KpCld is more susceptible to oxidative degradation under turnover conditions than the well-characterized Clds associated with perchlorate respiration. However, wild-type K. pneumoniae has a significant growth advantage in the presence of chlorate relative to a Δcld knockout strain, specifically under nitrate-respiring conditions. This suggests that a physiological function of KpCld may be detoxification of endogenously produced chlorite.

The heme-based chlorite dismutases catalyze the unimolecular decomposition of chlorite (ClO2 –) to yield Cl– and O2. The work presented here shows that chlorite dismutase from Dechloromonas aromatica (DaCld) also catalyzes the decomposition of bromite (BrO2 –) with the evolution of O2 (k cat = (2.0 ± 0.2) × 102 s–1; k cat /K M = (1.2 ± 0.2) × 105 M–1 s–1 at pH 5.2). Stopped-flow studies of this BrO2 – decomposition as a function of pH show that (1) the two-electron oxidized heme, compound I (Cpd I), is the primary accumulating heme intermediate during O2 evolution in acidic solution, (2) Cpd I and its one-electron reduction product, compound II (Cpd II), are present in varying ratios at intermediate pHs, and (3) only Cpd II is observed at pH 9.0. The pH dependences of Cpd I and Cpd II populations both yield a pK a of 6.7 ± 0.1 in good agreement with the pK a of DaCld activity with ClO2 –. The observation of a protein-based amino acid radical (AA•), whose appearance coincides with that of Cpd II, supports the hypothesis that the conversion of Cpd I to Cpd II occurs via proton-coupled electron transfer (PCET) from a heme-pocket amino acid to the oxidized porphyrinate of Cpd I to yield a dead-end decoupled state in which the holes decay at different rates. The site of the amino acid radical is tentatively assigned to Y118, which serves as a H-bond donor to propionate 6 (P6). The favoring of Cpd II:AA• accumulation in alkaline solution is consistent with the amino acid oxidation being rate limited by transfer of its proton to P6 having a pK a of 6.7. Examination of reaction mixtures comprising DaCld and ClO2 – by resonance Raman and electron paramagnetic resonance spectroscopy reveal the formation of Cpd II and •ClO2, which forms in preference to the analogous AA• in the BrO2 – reaction. The addition of ClO– to Cpd II did not yield O2. Together, these results are consistent with heterolytic cleavage of the O–BrO– and O–ClO– bonds yielding Cpd I, which is the catalytically active intermediate. The long-lived Cpd II that forms subsequently, is inactive toward O2 production, and diminishes the amount of enzyme available to cycle through the active Cpd I intermediate.

COVID-19 is a pandemic with high morbidity and mortality. In an autopsy cohort of COVID-19 patients, we found extensive accumulation of the tryptophan degradation products 3-hydroxy-anthranilic acid and quinolinic acid in the lungs, heart, and brain. This was not related to the expression of the tryptophan-catabolizing indoleamine 2,3-dioxygenase (IDO)-1, but rather to that of its isoform IDO-2, which otherwise is expressed rarely. Bioavailability of tryptophan is an absolute requirement for proper cell functioning and synthesis of hormones, whereas its degradation products can cause cell death. Markers of apoptosis and severe cellular stress were associated with IDO-2 expression in large areas of lung and heart tissue, whereas affected areas in brain were more restricted. Analyses of tissue, cerebrospinal fluid, and sequential plasma samples indicate early initiation of the kynurenine/ aryl-hydrocarbon receptor/IDO-2 axis as a positive feedback loop, potentially leading to severe COVID-19 pathology.

O2-evolving chlorite dismutases (Clds) fall into two subfamilies, which efficiently convert ClO2− to O2 and Cl−. The Cld from Dechloromonas aromatica (DaCld) represents the chlorite-decomposing homopentameric enzymes found in perchlorate and chlorate respiring bacteria. The Cld from the Gram-negative, human pathogen Klebsiella pneumoniae (KpCld) is representative of the second subfamily, comprising homodimeric enzymes having truncated N-termini. Here steric and nonbonding properties of the DaCld and KpCld active sites have been probed via kinetic, thermodynamic and spectroscopic behaviors of their fluorides, chlorides and hydroxides. Cooperative Cl− binding to KpCld drives formation of a hexacoordinate, high-spin aqua heme, whereas DaCld remains pentacoordinate and high-spin under analogous conditions. Fluoride coordinates to the heme iron in KpCld and DaCld, exhibiting ν(FeIII−F) bands at 385 and 390 cm−1, respectively. Correlation of these frequencies with their CT1 energies reveals strong H-bond-donation to the F− ligand, indicating that atoms directly coordinated to heme iron are accessible by distal H-bond donation. New vibrational frequency correlations between either ν(FeIII−F) or ν(FeIII−OH) and ν(FeII−His) of Clds and other heme proteins are reported. These correlations orthogonalize proximal and distal effects on the bonding between iron and exogenous π-donor ligands. The axial Fe−X vibrations and the relationships between them illuminate both similarities and differences in the H-bonding and electrostatic properties of the distal and proximal heme environments in pentameric and dimeric Clds. Moreover, they provide general insight into the structural basis of reactivity toward substrates in heme-dependent enzymes and their mechanistic intermediates, especially those containing the ferryl moiety.

O-evolving chlorite dismutases (Clds) efficiently convert chlorite (ClO) to O and Cl. Dechloromonas aromatica Cld ( DaCld) is a highly active chlorite-decomposing homopentameric enzyme, typical of Clds found in perchlorate- and chlorate-respiring bacteria. The Gram-negative, human pathogen Klebsiella pneumoniae contains a homodimeric Cld ( KpCld) that also decomposes ClO, albeit with an activity 10-fold lower and a turnover number lower than those of DaCld. The interactions between the distal pocket and heme ligand of the DaCld and KpCld active sites have been probed via kinetic, thermodynamic, and spectroscopic behaviors of their cyanide complexes for insight into active site characteristics that are deterministic for chlorite decomposition. At 4.7 × 10 M, the K for the KpCld-CN complex is 2 orders of magnitude smaller than that of DaCld-CN and indicates an affinity for CN that is greater than that of most heme proteins. The difference in CN affinity between Kp- and DaClds is predominantly due to differences in k. The kinetics of binding of cyanide to DaCld, DaCld(R183Q), and KpCld between pH 4 and 8.5 corroborate the importance of distal Arg183 and a p K of ∼7 in stabilizing complexes of anionic ligands, including the substrate. The Fe-C stretching and FeCN bending modes of the DaCld-CN (ν, 441 cm; δ, 396 cm) and KpCld-CN (ν, 441 cm; δ, 356 cm) complexes reveal differences in their FeCN angle, which suggest different distal pocket interactions with their bound cyanide. Conformational differences in their catalytic sites are also reported by the single ferrous KpCld carbonyl complex, which is in contrast to the two conformers observed for DaCld-CO.