Analysis of the electrochemistry of hemes with E(m)s spanning 800 mV

Abstract

The free energy of heme reduction in different proteins is found to vary over more than 18 kcal/mol. It is a challenge to determine how proteins manage to achieve this enormous range of E(m)s with a single type of redox cofactor. Proteins containing 141 unique hemes of a-, b-, and c-type, with bis-His, His-Met, and aquo-His ligation were calculated using Multi-Conformation Continuum Electrostatics (MCCE). The experimental E(m)s range over 800 mV from -350 mV in cytochrome c(3) to 450 mV in cytochrome c peroxidase (vs. SHE). The quantitative analysis of the factors that modulate heme electrochemistry includes the interactions of the heme with its ligands, the solvent, the protein backbone, and sidechains. MCCE calculated E(m)s are in good agreement with measured values. Using no free parameters the slope of the line comparing calculated and experimental E(m)s is 0.73 (R(2) = 0.90), showing the method accounts for 73% of the observed E(m) range. Adding a +160 mV correction to the His-Met c-type hemes yields a slope of 0.97 (R(2) = 0.93). With the correction 65% of the hemes have an absolute error smaller than 60 mV and 92% are within 120 mV. The overview of heme proteins with known structures and E(m)s shows both the lowest and highest potential hemes are c-type, whereas the b-type hemes are found in the middle E(m) range. In solution, bis-His ligation lowers the E(m) by approximately 205 mV relative to hemes with His-Met ligands. The bis-His, aquo-His, and His-Met ligated b-type hemes all cluster about E(m)s which are approximately 200 mV more positive in protein than in water. In contrast, the low potential bis-His c-type hemes are shifted little from in solution, whereas the high potential His-Met c-type hemes are raised by approximately 300 mV from solution. The analysis shows that no single type of interaction can be identified as the most important in setting heme electrochemistry in proteins. For example, the loss of solvation (reaction field) energy, which raises the E(m), has been suggested to be a major factor in tuning in situ E(m)s. However, the calculated solvation energy vs. experimental E(m) shows a slope of 0.2 and R(2) of 0.5 thus correlates weakly with E(m)s. All other individual interactions show even less correlation with E(m). However the sum of these terms does reproduce the range of observed E(m)s. Therefore, different proteins use different aspects of their structures to modulate the in situ heme electrochemistry. This study also shows that the calculated E(m)s are relatively insensitive to different heme partial charges and to the protein dielectric constant used in the simulation.

Figure 1

MCCE calculated vs. experimental redox potential (mV).– (a) All E_m,calc vs. E_m,_expt (mV). (b) Averaging E_m,_calc for all proteins with multiple structures available (mV). Error bars show standard deviation of calculated E_ms. A +160 mV offset is added to all calculated His-Met c hemes. The two black arrows pointing downwards indicate the shifts in E_m,cals when the propionic acids were forced to be fully ionized in Rps. viridis reaction centers and B. pasteurii cyt c₅₅₃ (See Results and Discussion). ◆ (green): bis-His a type. ◆ (blue): bis-His b type. ◆ (red): bis-His c type. ○ (blue): His-Met b type. ○ (red): His-Met c type. +(blue): aqua-His b. +(red): aqua-His c. Central black solid line is where experimental and calculated E_ms are equal. The dashed line in (a) is the 160 mV offset for His-Met c-type. The black dot dashed and double dot dashed lines are the ±60 and ±120 mV error lines. The arrows pointing to the y-axis show the E_m,sols used in this calculation, which are −220 mV for bis-His, −120 mV for His-water, −60 mV for bis-His a and −15 mV for His-Met. The range of previous heme E_ms calculations is also shown.,,

Figure 2

Energy terms contributing to the E_m,calc vs. E_m,expt. (a) ΔΔG_rxn: loss of reaction field energy (desolvation energy); pairwise interaction with (b) ΔG_pol: backbone dipoles; (c) ΔG_res,prop: propionic acids; (d) ΔG_res,prot: sidechains. Symbols are the same as in Figure 1. The slope and R² values for each figure can be found in Table I.

Figure 3

Residue backbone dipoles with strong interaction with the heme. (a) bovine b₅ 1CYO with a total ΔG_pol of 123 meV. (b) Cyt. c₅₅₀ 1MZ4 with a total ΔG_pol of −158 meV. Distances between the iron and the carbonyl group, amine are labeled in Å. The two axial Histidine ligands and one Proline adjacent to the ligand are shown.

Figure 4

MCCE Calculated E_ms with different charge distribution and dielectric constants. (a) (■) Calculated E_ms of His-Met hemes using quantum charge set from Autenrieth F and coworkers vs. metal centered charge set. Calculated E_ms of bis-His hemes using (◆) ESP and (◇) NBO charge sets vs. metal centered charge set. (b) (●) Calculated E_ms of selected proteins in ε = 8 vs. ε = 4. Two dashed lines are +60 mV error lines. (c) Calculated E_ms using both ε = 8 and ε = 4 vs. experimental E_ms. Here, a +160 mV offset is added to all calculated His-Met c hemes for better comparison with the experimental data (See Fig. 1). The dashed line and dark line are the best-fit linear regression for calculated E_ms with ε = 8 (slope 0.84, R² 0.89) and ε = 4 (slope 0.92, R² 0.92), respectively.