Spectroscopic Characterization of Site-Specific [Fe4S4] Cluster Chemistry in Ferredoxin:Thioredoxin Reductase: Implications for the Catalytic Mechanism

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• 作者：Elizabeth M. Walters ; Ricardo Garcia-Serres ; Guy N. L. Jameson ; Dominique A. Glauser ; Florence Bourquin ; Wanda Manieri ; Peter Schü ; rmann ; Michael K. Johnson ; and Boi Hanh Huynh
• 刊名：Journal of the American Chemical Society
• 出版年：2005
• 出版时间：July 6, 2005
• 年：2005
• 卷：127
• 期：26
• 页码：9612 - 9624
• 全文大小：297K
• 年卷期：v.127,no.26(July 6, 2005)
• ISSN：1520-5126

文摘

Light regulation of enzyme activities in oxygenic photosynthesis is mediated by ferredoxin:thioredoxin reductase (FTR), a novel class of disulfide reductase with an active site comprising a [Fe₄S₄]²⁺cluster and an adjacent disulfide, that catalyzes reduction of the thioredoxin disulfide in two sequentialone-electron steps using a [Fe₂S₂]^2+/+ ferredoxin as the electron donor. In this work, we report onspectroscopic (EPR, VTMCD, resonance Raman, and Mössbauer) and redox characterization of the activesite of FTR in various forms of the enzyme, including wild-type FTR, point-mutation variants at each of theactive-site cysteine residues, and stable analogues of the one-electron-reduced FTR-Trx heterodisulfideintermediate. The results reveal novel site-specific Fe₄S₄-cluster chemistry in oxidized, one-electron-reduced,and two-electron-reduced forms of FTR. In the resting enzyme, a weak interaction between the Fe₄S₄cluster and the active-site disulfide promotes charge buildup at a unique Fe site and primes the active siteto accept an electron from ferredoxin to break the disulfide bond. In one-electron-reduced analogues,cleavage of the active-site disulfide is accompanied by coordination of one of the cysteine residues thatform the active-site disulfide to yield a [Fe₄S₄]³⁺ cluster with two cysteinate ligands at a unique Fe site. Themost intriguing result is that two-electron-reduced FTR in which the disulfide is reduced to a dithiol containsan unprecedented electron-rich [Fe₄S₄]²⁺ cluster comprising both valence-delocalized and valence-localizedFe²⁺Fe³⁺ pairs. These results provide molecular level insights into the catalytic mechanism of FTR, andtwo viable mechanisms are proposed.