Abstract
Chlorite dismutases (Clds) are heme b containing oxidoreductases that convert chlorite to chloride and molecular oxygen. In order to elucidate the role of conserved heme cavity residues in the catalysis of this reaction comprehensive mutational and biochemical analyses of Cld from “Candidatus Nitrospira defluvii” (NdCld) were performed. Particularly, point mutations of the cavity-forming residues R173, K141, W145, W146, and E210 were performed. The effect of manipulation in 12 single and double mutants was probed by UV–vis spectroscopy, spectroelectrochemistry, pre-steady-state and steady-state kinetics, and X-ray crystallography. Resulting biochemical data are discussed with respect to the known crystal structure of wild-type NdCld and the variants R173A and R173K as well as the structures of R173E, W145V, W145F, and the R173Q/W146Y solved in this work. The findings allow a critical analysis of the role of these heme cavity residues in the reaction mechanism of chlorite degradation that is proposed to involve hypohalous acid as transient intermediate and formation of an O=O bond. The distal R173 is shown to be important (but not fully essential) for the reaction with chlorite, and, upon addition of cyanide, it acts as a proton acceptor in the formation of the resulting low-spin complex. The proximal H-bonding network including K141-E210-H160 keeps the enzyme in its ferric (E°′ = −113 mV) and mainly five-coordinated high-spin state and is very susceptible to perturbation.
Highlights
X-ray structures are available for representatives of both phylogenetically separated main Chlorite dismutases (Clds) lineages
The prosthetic group in chlorite dismutases is heme b, which is proximally coordinated by a histidine (His[160], NdCld numbering) that is hydrogen-bonded to a glutamate residue (E210)
To obtain plasmids for expressing the NdCld variants R173 by small hydrophobic alanine (R173A), R173K, R173E, R173Q, E210A, K141E, W145F, W145V, W146Y mutagenesis was carried out using PhusionFlash polymerase (Finnzymes) with primers and their reverse complements listed in Supplemental Table 1, Supporting Information
Summary
X-ray structures are available for representatives of both phylogenetically separated main Cld lineages. The prosthetic group in chlorite dismutases is heme b, which is proximally coordinated by a histidine (His[160], NdCld numbering) that is hydrogen-bonded to a glutamate residue (E210) This couple of amino acids, together with neighboring lysine (K141) and two tryptophan residues (W145, W146), are found in all structures of functional Clds solved so far (Figure 1).[5−8] The most prominent and fully conserved amino acid residue on the distal side of functional Clds is an arginine (R173). Two Clds have been used for those studies so far, namely, chlorite dismutases from Dechloromonas aromatica (DaCld)[11,17] and NdCld,[6] suggesting a catalytic role for the distal arginine[6,11] and the formation of protein radical(s).[17] Notably, DaCld and NdCld show tremendous spectral differences [e.g., the Soret maximum at pH 7.0 has been reported to be at 393 nm (DaCld)[17] and 408 nm (NdCld)10]. The importance of R173 in the chlorite degradation reaction and formation of the Cld-cyanide complex is demonstrated and discussed as is the role of the K141-E210H160 triad in maintenance of the heme cavity architecture and redox properties
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