| De novo heme protein design is a constructive and flexible method used to illustrate the structure-function relationships of natural heme proteins. In this work, the repertoire of axial ligands and heme types were expanded beyond the commonly used bis-histidine and heme b motif. In a designed four-alpha-helix bundle, [Delta7-H10I14 I21]2, the axial ligand histidine was systematically changed to seven different non-natural amino acids, 3-methyl-L-histidine (H3m), 1-methyl-L-histidine (H1m), 3-(1,2,4-triazol-1-yl)- L-alanine (Ntr), beta-(5-Tetrazolyl)-L-alanine (Ttr), beta-(4-pyridyl)- L-alanine (Pal4), beta-(3-pyridyl)-L-alanine (Pal3) and beta-(2-pyridyl)-L-alanine (Pal2). UV-visible, EPR, MCD and rR spectroscopies were used to examine the properties of these designed heme proteins. The data reveal that [Delta7-H3m10I14I 21]2_heme b is a spectroscopic model of cytochrome b5, a natural bis-His cytochrome. [Delta7-H1m 10I14I21]2_ferrous heme b is a spectroscopic model of deoxymyoglobin. [Delta7-Ntr10I 14I21]2 and [Delta7-Pal310I 14I21]2 only binds ferrous heme b and not ferric heme b. [Delta7-Pal410I 14I21]2, shows much weaker ferric heme b affinity than that of [Delta7-H3m10I14I21] 2, but its ferrous heme b affinity is much tighter than that of [Delta7-H3m10I14I21] 2. Finally, both [Delta7-Ttr10I14I21] 2 and [Delta7-Pal210I14I21] 2 scaffolds do not bind either ferric or ferrous heme b. The heme affinities and reduction potentials of the hemes were then investigated and explained in terms of sigma- and pi-bonding.; Inside two different heme protein scaffolds, [H10A24]2 and [Delta7-H3M10I14I21]2, the effect of porphyrin structure on heme affinity and electrochemistry were examined. Five hemes that have systematic changes on their periphery groups are used, which include three natural hemes, heme b, heme a and heme o, as well as two synthetic hemes, heme oh and Fe(dadpix). The electron-withdrawing groups on heme a and Fe(dadpix) contribute to the longer wavelength in the optical spectra and the higher midpoint potentials for these hemes in the protein scaffolds. The long farnesyl tail in heme a, heme oh and heme o lead to tight ferric heme affinities. The scaffolds [H10A24]2 and [Delta7-H3m10I14 I21]2 do not affect the optical spectra or ferric heme binding affinity. However, due to the better heme burial inside the large scaffold [H10A24]2, there is a consistently higher midpoint potential in this scaffold than that in the smaller one, [Delta7-H3m10I 14I21]2 for the same type of heme.; In conclusion, altering the axial ligands or the heme structure brings fresh steric and electronic properties that dramatically change the heme protein properties and functions and contribute to future design of tailored-made heme proteins. |
