Manipulating conserved heme cavity residues of chlorite dismutase: effect on structure, redox chemistry, and reactivity

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.

Figure 1

Overlay of subunit and distal heme architecture of NdCld wild-type and variants. (A) Cartoon representation of NdCld wild-type and the variants R173E, R173QW146Y, W145V, and W145F with the respective heme and active site residues represented as sticks. (B–D) Enlargement of the distal heme side of NdCld wild-type (B), and variants R173QW146Y (C) and R173E (D). Figures were generated using PyMOL (http://www.pymol.org/).

Figure 2

Architecture of the proximal heme side of wild-type NdCld (A) and the variants W146Y (B), W145V (C) and W145F together with its 2|F_o| – |F_c| electron density map countered at 1σ level (D). Figures were generated using PyMOL (http://www.pymol.org/). Putative H-bonds are shown as dotted black lines.

Figure 3

UV–vis spectra of NdCld wild-type and mutant proteins in oxidized (ferric) and reduced (ferrous) states at pH 7.0. (A) UV–vis spectra of distal side variants including the double mutant R173Q/W146Y. (B) UV–vis spectra of proximal side variants. Spectra of ferric proteins are depicted in black, and those of ferrous forms are in red.

Figure 4

Kinetics of cyanide binding to ferric high-spin NdCld Trp145Phe followed by stopped-flow spectroscopy. (A) Spectral changes upon reaction of 0.5 μM NdCld W145F with 10 μM cyanide measured in the conventional stopped-flow mode. The first spectrum shows native high-spin NdCld W145F (Soret band at 408 nm), the second spectrum was recorded 1.3 ms after mixing. Subsequent spectra show the formation of the low-spin cyanide complex (absorbance maximum 420 nm). Arrows indicate changes of absorbance with time. Conditions: 50 mM phosphate buffer, pH 7.0, and 25 °C. The inset shows a typical time trace at 408 nm with double exponential fit (0.7 μM NdCld W145F and 15 μM cyanide). Linear dependence of k_obs(1) and k_obs(2) (inset) from the cyanide concentration is shown in (B) for NdCld W145F (black), NdCld W145V (red), NdCld W146Y (green), NdCld W145V/W146Y (blue), as well as in (C) for NdCld R173E (black), NdCld R173Q (red), and NdCld R173Q/W146Y (green).

Figure 5

Spectroelectrochemistry of NdCld variants. Representative electronic spectra of (A) wild-type chlorite dismutase from “Candidatus Nitrospira defluvii”, (B) NdCld R173E, (C) NdCld R173K, and (D) NdCld W145F at various potentials at 25 °C and pH 7.0. The insets depict the corresponding Nernst plots, were X represents the ratio (A_λred^max – A_λred)/(A_λox^max – A_λox). For wild-type NdCld: λ_ox = 410 nm and λ_red = 435 nm. For the variants, the corresponding Soret maxima of the ferric and ferrous states are summarized in Table 3.

Figure 6

Reduction thermodynamics of wild-type NdCld and the variants R173K and W145F. (A) Temperature dependence of the reduction potential and (B) E°′/T versus 1/T plots for wild-type NdCld (circles), NdCld R173K (squares) and NdCld W145F (diamonds). The slope of the plot yields the ΔS_rc^°′/F (A) and −ΔH_rc^°′/F (B) values, respectively. Solid lines are least-squares fits to the data points. All experiments were carried out in 150 mM phosphate buffer, pH 7.0, containing 100 mM NaCl.