Rewiring the "Push-Pull" Catalytic Machinery of a Heme Enzyme Using an Expanded Genetic Code

Abstract

Nature employs a limited number of genetically encoded axial ligands to control diverse heme enzyme activities. Deciphering the functional significance of these ligands requires a quantitative understanding of how their electron-donating capabilities modulate the structures and reactivities of the iconic ferryl intermediates compounds I and II. However, probing these relationships experimentally has proven to be challenging as ligand substitutions accessible via conventional mutagenesis do not allow fine tuning of electron donation and typically abolish catalytic function. Here, we exploit engineered translation components to replace the histidine ligand of cytochrome c peroxidase (CcP) by a less electron-donating N _δ-methyl histidine (Me-His) with little effect on the enzyme structure. The rate of formation (k ₁) and the reactivity (k ₂) of compound I are unaffected by ligand substitution. In contrast, proton-coupled electron transfer to compound II (k ₃) is 10-fold slower in CcP Me-His, providing a direct link between electron donation and compound II reactivity, which can be explained by weaker electron donation from the Me-His ligand ("the push") affording an electron-deficient ferryl oxygen with reduced proton affinity ("the pull"). The deleterious effects of the Me-His ligand can be fully compensated by introducing a W51F mutation designed to increase "the pull" by removing a hydrogen bond to the ferryl oxygen. Analogous substitutions in ascorbate peroxidase lead to similar activity trends to those observed in CcP, suggesting that a common mechanistic strategy is employed by enzymes using distinct electron transfer pathways. Our study highlights how noncanonical active site substitutions can be used to directly probe and deconstruct highly evolved bioinorganic mechanisms.

Figure 1

Structure and catalytic mechanism of cytochrome c peroxidase (CcP). (a) Active site of CcP (PDB code: 1ZBY). The hydrogen bond between Asp235 and His175 imparts a degree of imidazolate-like character onto the axial ligand, thus increasing its electron-donating capabilities. (b) Catalytic cycle of CcP with its biological redox partner ferrous cytochrome c (cytc) as an electron donor. His52 is protonated in compound I but not the resting state.

Figure 2

Structural characterization of CcP His and CcP Me-His. Overlay of CcP (PDB code: 1ZBY) and CcP Me-His (PDB code: 6H08) active sites (using secondary structure superpose, RMS deviation of 0.107 Å). The CcP His heme cofactor and key residues are shown as atom colored sticks with gray carbons. The protein backbone is represented in cartoon format (colored in gray). The CcP Me-His heme cofactor and key residues are shown as atom colored sticks with pink carbons. The protein backbone is represented in cartoon format (colored in pink). The 2F_o–F_c electron density map corresponding to the Me-His residue is contoured at 1σ (blue mesh).

Figure 3

Kinetic and spectroscopic characterization of CcP His and CcP Me-His. (a) Michaelis–Menten plots of cytc oxidation by CcP His (black, k_cat = 805 ± 25 s^–1, K_M^cytc = 18 ± 3 μM) and CcP Me-His (red, k_cat = 38 ± 5 s^–1, K_M^cytc = 17 ± 2 μM). Measurements at pH 6, 25 °C, error bars are SEM, n = 3. (b) Overlay of the UV–vis spectra of the compound I states of CcP (black) and CcP Me-His (red).

Figure 4

X-band continuous wave EPR spectra of the compound I state of CcP His and CcP Me-His with and without the additional W51F mutation. EPR spectra recorded at 6 K showing the effect of tryptophan-(indole-d₅) on the spectra and a comparison with the CcP Me-His W191F variant; g values are marked, and red arrows indicate partially resolved hyperfine splitting.

Figure 5

Pre-steady-state kinetic characterization of CcP His and CcP Me-His. (a) Catalytic mechanism of CcP. (b) Observed rate constants for compound I formation at varying H₂O₂ concentrations. Representative kinetic traces at 20 μM H₂O₂ are shown (inset). A linear fit of k_obs versus [H₂O₂] was used to derive bimolecular rate constants of k₁ = (3.5 ± 0.7) × 10⁷ M^–1 s^–1 for CcP His (black) and k₁ = (3.3 ± 0.4) × 10⁷ M^–1 s^–1 for CcP Me-His (red). Error bars represent SD, n = 3. (c) Averaged kinetic traces (n = 3) for compound I reduction for both CcP His (black) and CcP Me-His (red). Conditions: CcP His/CcP Me-His (4 μM), H₂O₂ (8 μM), and a delay time of 1 s before reaction with cytc (1.5 μM) (post-mixing concentrations). Reactions were monitored by reduction in absorbance at 550 nm due to oxidation of ferrous cytc. These data are fitted to an integrated second-order rate equation (gray lines) to derive an apparent intrinsic rate constants of k₂ = (1.16 ± 0.12) × 10⁸ M^–1 s^–1 in CcP His and k₂ = (1.14 ± 0.25) × 10⁸ M^–1 s^–1 in CcP Me-His. The instrument dead time for the determination of k₂ is <1.5 ms. (d) Observed rate constants for compound II reduction at varying cytc concentrations. Representative kinetic traces at 35 μM are shown (inset), and all data were fitted to A₅₅₀ = A₀ + ΔA(e^{–k₂[cytc]₀t} + e^–k_obst) with k₂ fixed to the value determined above. A linear fit of k_obs versus [cytc] was used to derive bimolecular rate constants for CcP His (black, k₃ = (2.7 ± 0.2) × 10⁶ M^–1 s^–1) and CcP Me-His (red, k₃ = (2.6 ± 0.1 × 10⁵ M^–1 s^–1). Error bars are SD (n = 2 for CcP Me-His; n = 3–5 for CcP His). All measurements are at pH 6, 4 °C.

Figure 6

Solvent kinetic isotope effects and the role of Trp51 in controlling compound II proton affinity and reactivity. (a) Bar chart showing the solvent kinetic isotope effect on k_cat for cytc oxidation by CcP His and CcP Me-His at pH/pD = 6 and pH/pD = 5. CcP His: KSIE = 1.4 (pH/pD = 6) and KSIE = 2.2 (pH/pD = 5). CcP Me-His: KSIE = 1.5 (pH/pD = 6) and KSIE = 2.1 (pH/pD = 5). (b) Bar chart showing the kinetics (k_cat) of cytc oxidation by CcP His, CcP Me-His, and their W51F variants. (c–e) Schemes for proton-coupled compound II reduction in (c) CcP His, (d) CcP Me-His, and (e) CcP Me-His W51F, highlighting the importance of axial ligand electron donation (the “push”) and hydrogen-bonding interactions with Trp51 in controlling the proton affinity of the ferryl oxygen (the “pull”). The proton (“H”) is likely transferred from His52 via an ordered water molecule.

Figure 7

Kinetic characterization of ascorbate peroxidase (APX) and key active site mutants. (a) Active site of APX showing the substrate ascorbate bound at the γ-heme edge (PDB code: 1OAF). (b) Bar chart showing the kinetics (k_cat) of ascorbate oxidation by APX His, APX Me-His, and APX Me-His W51F. Measurements are recorded at 25 °C in phosphate buffer (50 mM, pH 6).