Spectroscopic studies of the tungsten-containing formaldehyde ferredoxin oxidoreductase from the hyperthermophilic archaeon Thermococcus litoralis

The electronic and redox properties of the iron-sulfur cluster and tungsten center in the as-isolated and sulfide-activated forms of formaldehyde ferredoxin oxidoreductase (FOR) from Thermococcus litoralis (Tl) have been investigated by using the combination of EPR and variable-temperature magnetic circular dichroism (VTMCD) spectroscopies. The results reveal a [Fe4S4]2+,+ cluster (E m = −368 mV) that undergoes redox cycling between an oxidized form with an S = 0 ground state and a reduced form that exists as a pH- and medium-dependent mixture of S = 3/2 (g = 5.4; E/D = 0.33) and S = 1/2 (g = 2.03, 1.93, 1.86) ground states, with the former dominating in the presence of 50% (v/v) glycerol. Three distinct types of W(V) EPR signals have been observed during dye-mediated redox titration of as-isolated Tl FOR. The initial resonance observed upon oxidation, termed the “low-potential” W(V) species (g = 1.977, 1.898, 1.843), corresponds to approximately 25–30% of the total W and undergoes redox cycling between W(IV)/ W(V) and W(V)/W(VI) states at physiologically relevant potentials (E m = − 335 and − 280 mV, respectively). At higher potentials a minor “mid-potential” W(V) species, g = 1.983, 1.956, 1.932, accounting for less than 5% of the total W, appears with a midpoint potential of − 34 mV and persists up to at least + 300 mV. At potentials above 0 mV, a major “high-potential ” W(V) signal, g = 1.981, 1.956, 1.883, accounting for 30–40% of the total W, appears at a midpoint potential of +184 mV. As-isolated samples of Tl FOR were found to undergo an approximately 8-fold enhancement in activity on incubation with excess Na2S under reducing conditions and the sulfide-activated Tl FOR was partially inactivated by cyanide. The spectroscopic and redox properties of the sulfideactivated Tl FOR are quite distinct from those of the as-isolated enzyme, with loss of the low-potential species and changes in both the mid-potential W(V) species (g=1.981, 1.950, 1.931; E m = − 265 mV) and highpotential W(V) species (g=1.981, 1.952, 1.895; E m=+ 65 mV). Taken together, the W(V) species in sulfideactivated samples of Tl FOR maximally account for only 15% of the total W. Both types of high-potential W(V) species were lost upon incubation with cyanide and the sulfide-activated high-potential species is converted into the as-isolated high-potential species upon exposure to air. Structural models are proposed for each of the observed W(V) species and both types of mid-potential and high-potential species are proposed to be artifacts of ligand-based oxidation of W(VI) species. A W(VI) species with terminal sulfido or thiol ligands is proposed to be responsible for the catalytic activity in sulfide-activated samples of Tl FOR.