Analysis of the electrochemistry of hemes with Ems spanning 800 mV

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 rcdox 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 cy-tochrome 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 approximate to 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 approximate to 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 approximate to 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.