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