Heme redox potential control in de novo designed four-alpha-helix bundle proteins.
The effects of various mechanisms of metalloporphyrin reduction potential modulation were investigated experimentally using a robust, well-characterized heme protein maquette, synthetic protein scaffold H10A24 [¿CH(3)()CONH-CGGGELWKL.HEELLKK.FEELLKL.AEERLKK. L-CONH(2)()¿(2)](2). Removal of the iron porphyrin macrocycle from the high dielectric aqueous environment and sequestration within the hydrophobic core of the H10A24 maquette raises the equilibrium reduction midpoint potential by 36-138 mV depending on the hydrophobicity of the metalloporphyrin structure. By incorporating various natural and synthetic metalloporphyrins into a single protein scaffold, we demonstrate a 300-mV range in reduction potential modulation due to the electron-donating/withdrawing character of the peripheral macrocycle substituents. Solution pH is used to modulate the metalloporphyrin reduction potential by 160 mV, regardless of the macrocycle architecture, by controlling the protonation state of the glutamate involved in partial charge compensation of the ferric heme. Attempts to control the reduction potential by inserting charged amino acids into the hydrophobic core at close proximity to the metalloporphyrin lead to varied success, with H10A24-L13E lowering the E(m8.5) by 40 mV, H10A24-E11Q raising it by 50 mV, and H10A24-L13R remaining surprisingly unaltered. Modifying the charge of the adjacent metalloporphyrin, +1 for iron(III) protoporphyrin IX or neutral for zinc(II) protoporphyrin IX resulted in a loss of 70 mV [Fe(III)PPIX](+) - [Fe(III)PPIX](+) interaction observed in maquettes. Using these factors in combination, we illustrate a 435-mV variation of the metalloporphyrin reduction midpoint potential in a simple heme maquette relative to the about 800-mV range observed for natural cytochromes. Comparison between the reduction potentials of the heme maquettes and other de novo designed heme proteins reveals global trends in the E(m) values of synthetic cytochromes.
Shifman
J M
JM
The Johnson Research Foundation, Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
Gibney
B R
BR
Sharp
R E
RE
Dutton
P L
PL
D000595:Amino Acid Sequence; D003580:Cytochromes / Q000737:chemistry; D006418:Heme / Q000737:chemistry*; D006420:Hemeproteins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011199:Potentiometry; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017186:Titrimetry
D003580:Cytochromes; D006420:Hemeproteins; D006418:Heme