Systematic Perturbations of Binuclear Non-heme Iron Sites: Structure and Dioxygen Reactivity of de Novo Due Ferri Proteins

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• 作者：Rae Ana Snyder ; Justine Betzu ; Susan E. Butch ; Amanda J. Reig ; William F. DeGrado ; Edward I. Solomon
• 刊名：Biochemistry
• 出版年：2015
• 出版时间：August 4, 2015
• 年：2015
• 卷：54
• 期：30
• 页码：4637-4651
• 全文大小：873K
• ISSN：1520-4995

文摘

DFsc (single-chain due ferri) proteins allow for modeling binuclear non-heme iron enzymes with a similar fold. Three 4A 鈫?4G variants of DFsc were studied to investigate the effects of (1) increasing the size of the substrate/solvent access channel (G4DFsc), (2) including an additional His residue in the first coordination sphere along with three additional helix-stabilizing mutations [3His-G4DFsc(Mut3)], and (3) the three helix-stabilizing mutations alone [G4DFsc(Mut3)] on the biferrous structures and their O₂ reactivities. Near-infrared circular dichroism and magnetic circular dichroism (MCD) spectroscopy show that the 4A 鈫?4G mutations increase coordination of the diiron site from 4-coordinate/5-coordinate to 5-coordinate/5-coordinate, likely reflecting increased solvent accessibility. While the three helix-stabilizing mutations [G4DFsc(Mut3)] do not affect the coordination number, addition of the third active site His residue [3His-G4DFsc(Mut3)] results in a 5-coordinate/6-coordinate site. Although all 4A鈫?4G variants have significantly slower pseudo-first-order rates when reacting with excess O₂ than DFsc (鈭? s^鈥?), G4DFsc and 3His-G4DFsc(Mut3) have rates (鈭?.02 and 鈭?.04 s^鈥?) faster than that of G4DFsc(Mut3) (鈭?.002 s^鈥?). These trends in the rate of O₂ reactivity correlate with exchange coupling between the Fe(II) sites and suggest that the two-electron reduction of O₂ occurs through end-on binding at one Fe(II) rather than through a peroxy-bridged intermediate. UV鈥搗is absorption and MCD spectroscopies indicate that an Fe(III)Fe(III)-OH species first forms in all three variants but converts into an Fe(III)-渭-OH-Fe(III) species only in the 2-His forms, a process inhibited by the additional active site His ligand that coordinatively saturates one of the iron centers in 3His-G4DFsc(Mut3).