The nitric-oxide reductase from Paracoccus denitrificans uses a single specific proton pathway

Abstract

The NO reductase from Paracoccus denitrificans reduces NO to N2O (2NO + 2H(+) + 2e(-) → N2O + H2O) with electrons donated by periplasmic cytochrome c (cytochrome c-dependent NO reductase; cNOR). cNORs are members of the heme-copper oxidase superfamily of integral membrane proteins, comprising the O2-reducing, proton-pumping respiratory enzymes. In contrast, although NO reduction is as exergonic as O2 reduction, there are no protons pumped in cNOR, and in addition, protons needed for NO reduction are derived from the periplasmic solution (no contribution to the electrochemical gradient is made). cNOR thus only needs to transport protons from the periplasm into the active site without the requirement to control the timing of opening and closing (gating) of proton pathways as is needed in a proton pump. Based on the crystal structure of a closely related cNOR and molecular dynamics simulations, several proton transfer pathways were suggested, and in principle, these could all be functional. In this work, we show that residues in one of the suggested pathways (denoted pathway 1) are sensitive to site-directed mutation, whereas residues in the other proposed pathways (pathways 2 and 3) could be exchanged without severe effects on turnover activity with either NO or O2. We further show that electron transfer during single-turnover reduction of O2 is limited by proton transfer and can thus be used to study alterations in proton transfer rates. The exchange of residues along pathway 1 showed specific slowing of this proton-coupled electron transfer as well as changes in its pH dependence. Our results indicate that only pathway 1 is used to transfer protons in cNOR.

Keywords: Bioenergetics/Electron Transfer Complex; Electron Transfer; Enzyme Kinetics; Flow-Flash; Heme-Copper Oxidase; Kinetic Isotope Effect; Membrane Biophysics; Nitric Oxide; Proton Transport.

FIGURE 1.

Structure of cNOR from P. aeruginosa (Protein Data Bank code 3O0R) (13) with the predicted proton transfer pathways. The NorB (light gray, transparent) and NorC (black, transparent in B–D) subunits are shown in a helical representation. A, the residues that are predicted to be involved in proton transfer are indicated with sticks, in cyan (pathway 1), magenta (pathway 2), or wheat (pathway 3). The location of the K-pathway (not present in cNOR but important in other HCuOs) is indicated in dark red. B–D, close-up of the proton transfer pathways. The blue arrows indicate the point of entrance for waters from the bulk. The predicted proton transfer pathways are indicated with stick representations in cyan (pathway 1; A), in magenta (pathway 2; B), or wheat (pathway 3; C). The corresponding residues in P. denitrificans cNOR are indicated in parenthesis in case they are different or if they were exchanged in this study. Residues in NorC are indicated with a C in superscript. This figure was prepared with PyMOL (Schrödinger, LLC, New York).

FIGURE 2.

The kinetic (deuterium) isotope effect on ETPT during the reaction between fully reduced cNOR and O₂. A, trace obtained at 430 nm of wild type cNOR in H₂O or D₂O at pH(*) 7.5, showing the change in absorbance (ΔA) over time (with the laser flash set at t = 0). The data were normalized to the CO_off step at t = 0 for the rapid time scale and to the amplitude of the ETPT (which varies slightly between experiments) for the longer time scale. The laser artifact at t = 0 has been removed for clarity. The CO_off reaction results in a rapid increase of absorbance, and O₂ binding (with τ of ∼40–50 μs) then results in a decrease. On the longer time scale, the ETPT is seen as a further negative ΔA (τ of ∼20–25 ms in H₂O). B, the rate of the ETPT in D₂O (blue) and H₂O (black) as a function of pH(*). The D₂O data were fitted to a pK_a* of 6.7 ± 0.1 and a k_H (maximal rate at low pH) of 66 ± 3 s⁻¹, and the H₂O data (which are from Ref. 8) were fitted to a pK_a of 6.6 ± 0.1 and a k_H of 244 ± 7 s⁻¹. mAU, milliabsorbance units.

FIGURE 3.

The reaction between fully reduced cNOR variants and O₂. The traces show the change in absorbance (ΔA) over time (with the laser flash set at t = 0) for cNOR wild type and the constructed variants. A–C, variants in proton transfer PW 2 and 3; D–F, variants in PW 1. Data were recorded at 430 nm (A and D), 420 nm (B and E), and 550 nm (C and F). The laser artifact at t = 0 has been removed for clarity. At 420 nm, the CO_off results in a rapid decrease in absorbance on the fast time scale, and the subsequent oxidation of the hemes (ETPT) results in a further slower negative ΔA. The amplitude of the ETPT varied slightly between experiments for both WT and variants and was normalized at 550 nm (reporting on the heme c) and 430 nm to the same ΔA for easier comparison of the rates. For the 420 nm trace and the shorter time scale at 430 nm, the CO_off step was used for normalization. The ΔA in the Asp-185 variants are only normalized to the CO_off step, because their ETPT ΔA values deviate from WT much more than the variation between measurements. A–C, WT (black trace), Q394M (blue trace), Q398L (green trace), and N47F (red trace). Gln-384 and Gln-398 are located in PW 2, and Asn-47 is in PW 3. D–F, wild type (black trace), E58Q^C (blue trace), K54A^C (red trace), D185E (green trace), D185N (gray trace), and D185A (yellow trace). mAU, milliabsorbance units.

FIGURE 4.

The pH dependence of the ETPT in cNOR wild type and variants. The rates of the ETPT for cNOR wild type and the constructed variants that were affected at pH 7.5 are plotted as a function of pH: wild type (black; circles for the data from Ref. and crosses for the data with the slightly altered purification protocol), K54A^C (red triangles), E58Q^C (blue diamonds). The WT data (from Ref. 8) were fitted to a pK_a of 6.61 ± 0.05 and a k_H of 244 ± 7 s⁻¹ (black line); K54A^C data were fitted to a pK_a = 6.4 ± 0.1 and a k_H = 247 ± 11 s⁻¹ (red line); and E58Q^C data were fitted to a pK_a = 5.8 ± 0.1 and a k_H = 49 ± 2 s⁻¹ (blue line). However, data points for K54A^C did not follow the fit around pH 7–7.5, possibly because the one-exponential fit is an oversimplification for this mutant. As a comparison, also plotted (as a dotted red line) is the diffusion rate, assuming k_diff of ∼2 × 10⁸ m⁻¹ s⁻¹ (see “Results” for details). The error bar at pH 7.0 for K54A^C indicates that at this pH the range of possible fits is rather large.

FIGURE 5.

The proton transfer pathway in cNOR as indicated in this work. The structure is the same as in Fig. 1 (cNOR from P. aeruginosa (Protein Data Bank code 3O0R) (13)) and rendered in the same way (except that for clarity a loop instead of helical representation is used) with the residues in the start of pathway 1 indicated with sticks in cyan (pathway 1) and the ones predicted to be involved in the continuation of the proton pathway to the active site in green. The suggested pathway is indicated with a blue (dotted) arrow. Crystallographic waters in or around the pathway are indicated with blue crosses. The black dotted lines indicate hydrogen bonds. The yellow A and D indicate the heme b₃ propionate A and D, respectively.