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