Functional and protective hole hopping in metalloenzymes

Abstract

Electrons can tunnel through proteins in microseconds with a modest release of free energy over distances in the 15 to 20 Å range. To span greater distances, or to move faster, multiple charge transfers (hops) are required. When one of the reactants is a strong oxidant, it is convenient to consider the movement of a positively charged "hole" in a direction opposite to that of the electron. Hole hopping along chains of tryptophan (Trp) and tyrosine (Tyr) residues is a critical function in several metalloenzymes that generate high-potential intermediates by reactions with O₂ or H₂O₂, or by activation with visible light. Examination of the protein structural database revealed that Tyr/Trp chains are common protein structural elements, particularly among enzymes that react with O₂ and H₂O₂. In many cases these chains may serve a protective role in metalloenzymes by deactivating high-potential reactive intermediates formed in uncoupled catalytic turnover.

Fig. 1. (A) Driving-force optimized (−ΔG° = λ) electron tunneling timetable for Ru-modified proteins: azurin (black); cytochrome c (blue); cytochrome c-b562 (cyan); myoglobin (magenta); high-potential iron protein (red). The solid line shows an exponential dependence on metal center-to-center distance with decay factor β = 1.1 Å⁻¹. (B) When −ΔG° ≠ λ electron tunneling times (τ_et) increase above their minimum values (τ_min). The solid curve illustrates the tunneling-distance change (Δr) that increases the tunneling time by the same factor as the deviation of −ΔG° from λ (calculated with λ = 0.8 eV and β = 1.1 Å⁻¹). Equivalently, Δr is the reduction in tunneling distance necessary to maintain a driving-force-optimized tunneling time.

Fig. 2. Structural model of the functional radical transfer pathway (Y122-W48-Y356-Y731-Y730-C439) in E. coli RNR (PDB ID 6W4X). Surrounding Trp and Tyr residues within 10 Å of pathway residues are shown along with shortest edge–edge distances (Å, blue numbers).

Fig. 3. Network of Tyr and Trp residues in branched chains (10 Å maximum contact distance) connected to the E. coli RNR radical transfer pathway residues (βTyr122-βTrp48-βTyr356-αTyr731-αTyr730-αCys439, red). Distances (Å, blue numbers) are from the cryo-EM structure of the holo-enzyme (PDB ID 6W4X). Residue βTYR122 was 2,3,5-trifluoro-tyrosine in the structure.

Fig. 4. Structural model (PDB ID 1DNP) of the functional hole transfer pathway (*Fla-W382-W359-W306) in E. coli DNA photolyase. Surrounding Trp (magenta) and Tyr (red) residues within 7.5 Å of pathway residues are shown along with time constants for on-pathway (blue) and off-pathway (red) hole transfers.

Fig. 5. Network of Tyr and Trp residues in branched chains (7.5 Å maximum contact distance) connected to the E. coli DNA photolyase radical transfer pathway residues (FAD472-Trp382-Trp359-Trp306, red). Distances (Å, blue numbers) are from the X-ray crystal structure of the enzyme (PDB ID 1DNP).

Fig. 6. Network of Tyr and Trp residues in branched chains (10 Å maximum contact distance) connected to the yeast CCP hole transfer pathway residues (HEME-Trp191, red). Distances (Å, blue numbers) are from the X-ray crystal structure of the enzyme (PDB ID 2CYP). Residues with >20% sidechain solvent exposure are indicated with an asterisk.

Fig. 7. Network of Tyr and Trp residues in branched chains (10 Å maximum contact distance) connected to the hemes in (A) P450cam (PDB ID 1PHC), (B) CYP119 (1F4U), (C) CYP158 (1S1F), and (D) P450_BM3 (2IJ2). Distances (Å) between residues appear as blue numbers. Solvent-exposed residues are labeled with an asterisk.

Fig. 8. The Tyr/Trp hole migration pathway (10 Å contact distance) from the CYP3A4 (PDB ID 1TQN) heme to the enzyme surface is composed of just two residues: Trp126, Tyr99. The colormap illustrates the driving-force dependence of the kinetics modeling for this pathway. If formation of Trp126˙⁺ is endergonic by 100 meV and subsequent hole transfer to Tyr99 is exergonic by 200 meV, the CI survival time (τ_hop) is estimated to be 0.3 μs. τ_hop is given by the integral of the normalized CI decay curve.

Fig. 9. Network of Tyr and Trp residues in branched chains (7.5 Å maximum contact distance) connected to the FMN-Fe₂ ET pathway residues (red) in the FDP from Methanothermobacter marburgensis. Distances (Å, blue numbers) are from the X-ray crystal structure of the enzyme (PDB ID 2OHH). Previously identified chains are highlighted in green. Solvent-exposed residues are labeled with an asterisk.