Abstract
O(2)-evolving chlorite dismutases (Clds) efficiently convert chlorite (ClO(2)(-)) to O(2) 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(2)(-), 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(-9) M, the K(D) 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(off). 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(a) of ∼7 in stabilizing complexes of anionic ligands, including the substrate. The Fe-C stretching and FeCN bending modes of the DaCld-CN(-) (ν(Fe-C), 441 cm(-1); δ(FeCN), 396 cm(-1)) and KpCld-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 KpCld carbonyl complex, which is in contrast to the two conformers observed for DaCld-CO.