Role of Histidine-86 in the Catalytic Mechanism of Ferredoxin: Thioredoxin Reductase

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• 作者：Elizabeth M. Walters ; Ricardo Garcia-Serres ; Sunil G. Naik ; Florence Bourquin ; Dominique A. Glauser ; Peter Schrmann ; Boi Hanh Huynh ; Michael K. Johnson
• 刊名：Biochemistry
• 出版年：2009
• 出版时间：February 10, 2009
• 年：2009
• 卷：48
• 期：5
• 页码：1016-1024
• 全文大小：238K
• 年卷期：v.48,no.5(February 10, 2009)
• ISSN：1520-4995

文摘

Ferredoxin:thioredoxin reductase catalyzes the reduction of thioredoxins in plant chloroplasts using the [Fe₂S₂] ferredoxin as a one-electron donor and as such plays a central role in light regulation of oxygenic photosynthesis. The active-site comprises a [Fe₄S₄] cluster next to a redox-active disulfide that is cleaved in sequential one-electron steps and the combination of spectroscopic and crystallographic studies have revealed a catalytic mechanism involving novel site specific cluster chemistry in the oxidized, one-electron- and two-electron-reduced redox states. Histidine-86 has emerged as a potential proton donor/acceptor in the catalytic mechanism based on redox-related changes in the positioning of the imidazole ring during redox cycling and greatly decreased activity for the H86Y variant. Here we report on spectroscopic and redox characterization of the [Fe₄S₄] center in Synechocystis sp. PCC 6803 H86Y ferredoxin:thoredoxin reductase in the accessible redox states of both the as purified and N-ethylmaleimide-modified forms, using the combination of UV−visible absorption and variable-temperature magnetic circular dichroism, EPR, resonance Raman and Mssbauer spectroscopies. The results demonstrate that His86 is required for formation of the partially valence-localized [Fe₄S₄]²⁺ cluster that is the hallmark of two-electron-reduced intermediate. Taken together with the available structural data, the spectroscopic results indicate a functional role for His86 in protonation/deprotonation of the cluster-interacting thiol and anchoring the cluster interacting thiol in close proximity to the cluster in the two-electron-reduced intermediate.