title: Spectroscopic studies of the tungsten-containing formaldehyde ferredoxin oxidoreductase from the hyperthermophilic archaeon Thermococcus litoralis.
abstract: 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 (Em=-368mV) 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 (Em= -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 sulfide-activated 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; Em = -265 mV) and high-potential W(V) species (g=1.981, 1.952, 1.895; Em = +65 mV). Taken together, the W(V) species in sulfide-activated 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.
authors.affiliation: Department of Biochemistry & Molecular Biology and Center for Metalloenzyme Studies, University of Georgia, Athens 30602, USA.
authors.forename: I K
authors.initials: IK
authors.lastname: Dhawan
authors.forename: R
authors.initials: R
authors.lastname: Roy
authors.forename: B P
authors.initials: BP
authors.lastname: Koehler
authors.forename: S
authors.initials: S
authors.lastname: Mukund
authors.forename: M W
authors.initials: MW
authors.lastname: Adams
authors.forename: M K
authors.initials: MK
authors.lastname: Johnson
mesh_terms: D000445:Aldehyde Oxidoreductases / Q000737:chemistry* / Q000302:isolation & purification; D001105:Archaea / Q000201:enzymology*; D002384:Catalysis; D002942:Circular Dichroism; D003486:Cyanides / Q000032:analysis; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004795:Enzyme Stability; D007501:Iron / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D008961:Models, Structural; D010084:Oxidation-Reduction; D013440:Sulfides / Q000032:analysis; D014414:Tungsten / Q000737:chemistry* / Q000378:metabolism
chemical_list: D003486:Cyanides; D007506:Iron-Sulfur Proteins; D013440:Sulfides; D007501:Iron; D000445:Aldehyde Oxidoreductases; C080011:formaldehyde ferredoxin oxidoreductase; D014414:Tungsten