How pH Modulates the Reactivity and Selectivity of a Siderophore-Associated Flavin Monooxygenase

Flavin-containing monooxygenases (FMOs) catalyze the oxygenation of diverse organic molecules using O₂, NADPH, and the flavin adenine dinucleotide (FAD) cofactor. The fungal FMO SidA initiates peptidic siderophore biosynthesis via the highly selective hydroxylation of l-ornithine, while the related amino acid l-lysine is a potent effector of reaction uncoupling to generate H₂O₂. We hypothesized that protonation states could critically influence both substrate-selective hydroxylation and H₂O₂ release, and therefore undertook a study of SidA’s pH-dependent reaction kinetics. Consistent with other FMOs that stabilize a C4a-OO­(H) intermediate, SidA’s reductive half reaction is pH independent. The rate constant for the formation of the reactive C4a-OO­(H) intermediate from reduced SidA and O₂ is likewise independent of pH. However, the rate constants for C4a-OO­(H) reactions, either to eliminate H₂O₂ or to hydroxylate l-Orn, were strongly pH-dependent and influenced by the nature of the bound amino acid. Solvent kinetic isotope effects of 6.6 ± 0.3 and 1.9 ± 0.2 were measured for the C4a-OOH/H₂O₂ conversion in the presence and absence of l-Lys, respectively. A model is proposed in which l-Lys accelerates H₂O₂ release via an acid–base mechanism and where side-chain position determines whether H₂O₂ or the hydroxylation product is observed

Active Sites of O₂‑Evolving Chlorite Dismutases Probed by Halides and Hydroxides and New Iron–Ligand Vibrational Correlations

O₂-evolving chlorite dismutases (Clds) fall into two subfamilies, which efficiently convert ClO₂^– to O₂ and Cl^–. The Cld from <i>Dechloromonas aromatica</i> (<i>Da</i>Cld) represents the chlorite-decomposing homopentameric enzymes found in perchlorate- and chlorate-respiring bacteria. The Cld from the Gram-negative human pathogen <i>Klebsiella pneumoniae</i> (<i>Kp</i>Cld) is representative of the second subfamily, comprising homodimeric enzymes having truncated N-termini. Here steric and nonbonding properties of the <i>Da</i>Cld and <i>Kp</i>Cld active sites have been probed via kinetic, thermodynamic, and spectroscopic behaviors of their fluorides, chlorides, and hydroxides. Cooperative binding of Cl^– to <i>Kp</i>Cld drives formation of a hexacoordinate, high-spin aqua heme, whereas <i>Da</i>Cld remains pentacoordinate and high-spin under analogous conditions. Fluoride coordinates to the heme iron in <i>Kp</i>Cld and <i>Da</i>Cld, exhibiting ν­(Fe^III–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 to distal H-bond donation. New vibrational frequency correlations between either ν­(Fe^III–F) or ν­(Fe^III–OH) and ν­(Fe^II–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

Understanding How the Distal Environment Directs Reactivity in Chlorite Dismutase: Spectroscopy and Reactivity of Arg183 Mutants

The chlorite dismutase from <i>Dechloromonas aromatica</i> (<i>Da</i>Cld) catalyzes the highly efficient decomposition of chlorite to O₂ and chloride. Spectroscopic, equilibrium thermodynamic, and kinetic measurements have indicated that Cld has two pH sensitive moieties; one is the heme, and Arg183 in the distal heme pocket has been hypothesized to be the second. This active site residue has been examined by site-directed mutagenesis to understand the roles of positive charge and hydrogen bonding in O–O bond formation. Three Cld mutants, Arg183 to Lys (R183K), Arg183 to Gln (R183Q), and Arg183 to Ala (R183A), were investigated to determine their respective contributions to the decomposition of chlorite ion, the spin state and coordination states of their ferric and ferrous forms, their cyanide and imidazole binding affinities, and their reduction potentials. UV–visible and resonance Raman spectroscopies showed that <i>Da</i>Cld­(R183A) contains five-coordinate high-spin (5cHS) heme, the <i>Da</i>Cld­(R183Q) heme is a mixture of five-coordinate and six-coordinate high spin (5c/6cHS) heme, and <i>Da</i>Cld­(R183K) contains six-coordinate low-spin (6cLS) heme. In contrast to wild-type (WT) Cld, which exhibits p<i>K</i>_a values of 6.5 and 8.7, all three ferric mutants exhibited pH-independent spectroscopic signatures and kinetic behaviors. Steady state kinetic parameters of the chlorite decomposition reaction catalyzed by the mutants suggest that in WT <i>Da</i>Cld the p<i>K</i>_a of 6.5 corresponds to a change in the availability of positive charge from the guanidinium group of Arg183 to the heme site. This could be due to either direct acid–base chemistry at the Arg183 side chain or a flexible Arg183 side chain that can access various orientations. Current evidence is most consistent with a conformational adjustment of Arg183. A properly oriented Arg183 is critical for the stabilization of anions in the distal pocket and for efficient catalysis

Distinguishing Active Site Characteristics of Chlorite Dismutases with Their Cyanide Complexes

O₂-evolving chlorite dismutases (Clds) efficiently convert chlorite (ClO₂^–) to O₂ and Cl^–. <i>Dechloromonas aromatica</i> Cld (<i>Da</i>Cld) is a highly active chlorite-decomposing homopentameric enzyme, typical of Clds found in perchlorate- and chlorate-respiring bacteria. The Gram-negative, human pathogen <i>Klebsiella pneumoniae</i> contains a homodimeric Cld (<i>Kp</i>Cld) that also decomposes ClO₂^–, albeit with an activity 10-fold lower and a turnover number lower than those of <i>Da</i>Cld. The interactions between the distal pocket and heme ligand of the <i>Da</i>Cld and <i>Kp</i>Cld 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^–9 M, the <i>K</i>_D for the <i>Kp</i>Cld–CN^– complex is 2 orders of magnitude smaller than that of <i>Da</i>Cld–CN^– and indicates an affinity for CN^– that is greater than that of most heme proteins. The difference in CN^– affinity between <i>Kp</i>- and <i>Da</i>Clds is predominantly due to differences in <i>k</i>_off. The kinetics of binding of cyanide to <i>Da</i>Cld, <i>Da</i>Cld­(R183Q), and <i>Kp</i>Cld between pH 4 and 8.5 corroborate the importance of distal Arg183 and a p<i>K</i>_a of ∼7 in stabilizing complexes of anionic ligands, including the substrate. The Fe–C stretching and FeCN bending modes of the <i>Da</i>Cld–CN^– (ν_Fe–C, 441 cm^–1; δ_FeCN, 396 cm^–1) and <i>Kp</i>Cld–CN^– (ν_Fe–C, 441 cm^–1; δ_FeCN, 356 cm^–1) 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 <i>Kp</i>Cld carbonyl complex, which is in contrast to the two conformers observed for <i>Da</i>Cld–CO

Unusual Peroxide-Dependent, Heme-Transforming Reaction Catalyzed by HemQ

A recently proposed pathway for heme <i>b</i> biosynthesis, common to diverse bacteria, has the conversion of two of the four propionates on coproheme III to vinyl groups as its final step. This reaction is catalyzed in a cofactor-independent, H₂O₂-dependent manner by the enzyme HemQ. Using the HemQ from <i>Staphylococcus aureus</i> (<i>Sa</i>HemQ), the initial decarboxylation step was observed to rapidly and obligately yield the three-propionate harderoheme isomer III as the intermediate, while the slower second decarboxylation appeared to control the overall rate. Both synthetic harderoheme isomers III and IV reacted when bound to HemQ, the former more slowly than the latter. While H₂O₂ is the assumed biological oxidant, either H₂O₂ or peracetic acid yielded the same intermediates and products, though amounts significantly greater than the expected 2 equiv were required in both cases and peracetic acid reacted faster. The ability of peracetic acid to substitute for H₂O₂ suggests that, despite the lack of catalytic residues conventionally present in heme peroxidase active sites, reaction pathways involving high-valent iron intermediates cannot be ruled out

Structure-Based Mechanism for Oxidative Decarboxylation Reactions Mediated by Amino Acids and Heme Propionates in Coproheme Decarboxylase (HemQ)

Coproheme decarboxylase catalyzes two sequential oxidative decarboxylations with H₂O₂ as the oxidant, coproheme III as substrate and cofactor, and heme <i>b</i> as the product. Each reaction breaks a CC bond and results in net loss of hydride, via steps that are not clear. Solution and solid-state structural characterization of the protein in complex with a substrate analog revealed a highly unconventional H₂O₂-activating distal environment with the reactive propionic acids (2 and 4) on the opposite side of the porphyrin plane. This suggested that, in contrast to direct CH bond cleavage catalyzed by a high-valent iron intermediate, the coproheme oxidations must occur through mediating amino acid residues. A tyrosine that hydrogen bonds to propionate 2 in a position analogous to the substrate in ascorbate peroxidase is essential for both decarboxylations, while a lysine that salt bridges to propionate 4 is required solely for the second. A mechanism is proposed in which propionate 2 relays an oxidizing equivalent from a coproheme compound I intermediate to the reactive deprotonated tyrosine, forming Tyr^•. This residue then abstracts a net hydrogen atom (H^•) from propionate 2, followed by migration of the unpaired propionyl electron to the coproheme iron to yield the ferric harderoheme and CO₂ products. A similar pathway is proposed for decarboxylation of propionate 4, but with a lysine residue as an essential proton shuttle. The proposed reaction suggests an extended relay of heme-mediated e^–/H⁺ transfers and a novel route for the conversion of carboxylic acids to alkenes

A Dimeric Chlorite Dismutase Exhibits O₂‑Generating Activity and Acts as a Chlorite Antioxidant in <i>Klebsiella pneumoniae</i> MGH 78578

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 <i>Klebsiella pneumoniae</i> (<i>Kp</i>Cld) 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 <i>Kp</i>Cld, like others in its subfamily, should be able to make O₂ and may serve an <i>in vivo</i> 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. <i>Kp</i>Cld is more susceptible to oxidative degradation under turnover conditions than the well-characterized Clds associated with perchlorate respiration. However, wild-type <i>K. pneumoniae</i> has a significant growth advantage in the presence of chlorate relative to a Δ<i>cld</i> knockout strain, specifically under nitrate-respiring conditions. This suggests that a physiological function of <i>Kp</i>Cld may be detoxification of endogenously produced chlorite

O₂-Evolving Chlorite Dismutase as a Tool for Studying O₂-Utilizing Enzymes

The direct interrogation of fleeting intermediates by rapid-mixing kinetic methods has significantly advanced our understanding of enzymes that utilize dioxygen. The gas’s modest aqueous solubility (<2 mM at 1 atm) presents a technical challenge to this approach, because it limits the rate of formation and extent of accumulation of intermediates. This challenge can be overcome by use of the heme enzyme chlorite dismutase (Cld) for the rapid, <i>in situ</i> generation of O₂ at concentrations far exceeding 2 mM. This method was used to define the O₂ concentration dependence of the reaction of the class Ic ribonucleotide reductase (RNR) from <i>Chlamydia trachomatis</i>, in which the enzyme’s Mn^IV/Fe^III cofactor forms from a Mn^II/Fe^II complex and O₂ via a Mn^IV/Fe^IV intermediate, at effective O₂ concentrations as high as ∼10 mM. With a more soluble receptor, myoglobin, an O₂ adduct accumulated to a concentration of >6 mM in <15 ms. Finally, the C–H-bond-cleaving Fe^IV–oxo complex, <b>J</b>, in taurine:α-ketoglutarate dioxygenase and superoxo–Fe₂^III/III complex, <b>G</b>, in <i>myo</i>-inositol oxygenase, and the tyrosyl-radical-generating Fe₂^III/IV intermediate, <b>X</b>, in <i>Escherichia coli</i> RNR, were all accumulated to yields more than twice those previously attained. This means of <i>in situ</i> O₂ evolution permits a >5 mM “pulse” of O₂ to be generated in <1 ms at the easily accessible Cld concentration of 50 μM. It should therefore significantly extend the range of kinetic and spectroscopic experiments that can routinely be undertaken in the study of these enzymes and could also facilitate resolution of mechanistic pathways in cases of either sluggish or thermodynamically unfavorable O₂ addition steps