Role of Histidine-86 in the Catalytic Mechanism of Ferredoxin: Thioredoxin Reductase
Walters, Elizabeth M.
Naik, Sunil G.
Glauser, Dominique A.
Huynh, Boi Hanh
Johnson, Michael K.
Date de parution
Biochemistry, American Chemical Society (ACS), 2009/48/5/1016-1024
Ferredoxin:thioredoxin reductase catalyzes the reduction of thioredoxins in plant chloroplasts using the [Fe<sub>2</sub>S<sub>2</sub>] ferredoxin as a one-electron donor and as such plays a central role in light regulation of oxygenic photosynthesis. The active-site comprises a [Fe<sub>4</sub>S<sub>4</sub>] 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 [Fe<sub>4</sub>S<sub>4</sub>] center in <i>Synechocystis</i> 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 Mössbauer spectroscopies. The results demonstrate that His86 is required for formation of the partially valence-localized [Fe<sub>4</sub>S</sub>4</sub>]<sup>2+</sup> 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.
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Resource Types::text::journal::journal article
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