Heme Redox Potential Control in de Novo Designed Four--Helix Bundle Proteins

详细信息    查看全文

• 作者：Julia M. Shifman ; Brian R. Gibney ; R. Eryl Sharp ; and P. Leslie Dutton
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：December 5, 2000
• 年：2000
• 卷：39
• 期：48
• 页码：14813 - 14821
• 全文大小：126K
• 年卷期：v.39,no.48(December 5, 2000)
• ISSN：1520-4995

文摘

The effects of various mechanisms of metalloporphyrin reduction potential modulation wereinvestigated experimentally using a robust, well-characterized heme protein maquette, synthetic proteinscaffold H10A24 [{CH₃CONH-CGGGELWKL·HEELLKK·FEELLKL·AEERLKK·L-CONH₂}₂]₂. Removal of the iron porphyrin macrocycle from the high dielectric aqueous environment and sequestrationwithin the hydrophobic core of the H10A24 maquette raises the equilibrium reduction midpoint potentialby 36-138 mV depending on the hydrophobicity of the metalloporphyrin structure. By incorporatingvarious natural and synthetic metalloporphyrins into a single protein scaffold, we demonstrate a 300-mVrange in reduction potential modulation due to the electron-donating/withdrawing character of the peripheralmacrocycle substituents. Solution pH is used to modulate the metalloporphyrin reduction potential by160 mV, regardless of the macrocycle architecture, by controlling the protonation state of the glutamateinvolved in partial charge compensation of the ferric heme. Attempts to control the reduction potential byinserting charged amino acids into the hydrophobic core at close proximity to the metalloporphyrin leadto 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 hememaquette relative to the about 800-mV range observed for natural cytochromes. Comparison between thereduction potentials of the heme maquettes and other de novo designed heme proteins reveals globaltrends in the E_m values of synthetic cytochromes.