PARENT SESSION

Symposium S5B Light, redox and metabolic regulation: Dark reactions
Wednesday September 1st, 2004 10:20 AM-12:20 PM Room 510B
Chair: Peter Schürmann
Co-Chair: Toru Hisabori

Disulfide reduction mediated by site-specific [4Fe-4S] cluster chemistry. Michael Johnson^*,1, Elizabeth Waters¹, Guy Jameson², Boi Hanh Huyn², Peter Schurmann³, ¹ Department of Chemistry, Athens, Georgia, USA² Department of Physics, Atlantic, Georgia, USA³ Laboratoire de Biochemie Végétale, Neuchâtel, Switzerland

ABSTRACT- Thioredoxin-mediated light regulation in plant chloroplasts involves a unique class of disulfide reductases that catalyze disulfide reduction in two one-electron steps using a [2Fe-2S] ferredoxin as the electron donor and an active site comprising a [4Fe-4S] cluster and a redox-active disulfide. Spectroscopic studies of ferredoxin:thioredoxin reductase (FTR) from spinach and Synechocystis have identified a S = 0 [4Fe-4S]2+ cluster in both the as prepared (disulfide-intact) and two-electron-reduced (disulfide-cleaved) forms. However, freeze-quench EPR studies have identified a transient, slow-relaxing resonance, g = 2.11, 2.00, 1.98, corresponding to a one-electron-reduced catalytic intermediate. Stable analogs of this intermediate with identical EPR characteristics have been prepared by selectively modifying (NEM FTR) or mutating (C57A FTR) the cysteine of the active-site disulfide that forms a heterodisulfide intermediate with the thioredoxin substrate. Detailed spectroscopic characterization of NEM FTR using absorption, EPR, ENDOR, VTMCD, resonance Raman and Mössbauer spectroscopies indicates that the one-electron reduced catalytic intermediate involves two-electron disulfide reduction coupled with one-electron cluster oxidation to yield a unique type of S = 1/2 [4Fe-4S]3+ cluster with two cysteine residues ligated at a specific Fe site. Moreover, Mössbauer studies show that the one-electron oxidation of the cluster occurs predominantly at the unique Fe site. The results indicate a novel mechanism for disulfide cleavage in two one-electron steps involving site-specific cluster chemistry. A similar mechanism is proposed for direct [4Fe-4S]-mediated cleavage of the CoM-S-S-CoB heterodisulfide in methanogenic archaea by heterodisulfide reductases.

KEY WORDS: thioredoxin