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 (T1) 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 T1 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 T1 FOR were found to undergo an approximately 8-fold enhancement in activity on incubation with excess Na₂S under reducing conditions and the sulfide- activated T1 FOR was partially inactivated by cyanide. The spectroscopic and redox properties of the sulfide-activated T1 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 high-potential W(V) species (g = 1.981, 1.952, 1.895; E(m) = + 65 mV). Taken together, the W(V) species in sulfide-activated samples of T1 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 T1 FOR.