Spectroscopic characterization of the tungsten and iron centers in aldehyde ferredoxin oxidoreductases from two hyperthermophilic archaea

The electronic and redox properties of the iron and tungsten centers in the aldehyde ferredoxin oxidoreductases (AORs) from  Pyrococcusfuriosus  (Pf) and  Pyrococcus  strain ES-4 (ES-4) have been investigated by the combination of EPR and variable-temperature magnetic circular dichroism (VTMCD) spectroscopies. Parallel- and perpendicular-mode EPR studies of ES-4 AOR reveal a redox inactive “ g  = 16” resonance from an integer spin paramagnet. On the basis of the X-ray crystal structure of Pf AOR (Chan, M. K.; Mukund, S.; Kletzin, A.; Adams, M. W. W.; Rees, D. C.  Science 1995,  267 , 1463−1469), this resonance is attributed to a mononuclear high-spin Fe(II) ion at the subunit interface, although the possibility that this center is a carboxylate-bridged reduced diiron center in ES-4 AOR is also considered. Both enzymes have a [4Fe−4S] 2+,+  cluster with unique electronic properties compared to known synthetic or biological [4Fe−4S] +  clusters, i.e. pure  S  =  3 / 2  ground state with  g  = 4.7, 3.4, 1.9 ( E / D  = 0.12 and  D  = +4 cm -1 ). Seven distinct W(V) EPR signals have been observed during dye-mediated redox titrations of Pf AOR, and the four major W(V) species have been rigorously identified and characterized via EPR spectral simulations of natural abundance and  183 W-enriched samples ( 183 W,  I  =  1 / 2 , 14.28% natural abundance). Both enzymes contain two major forms of W, each corresponding to approximately 20−30% of the total W. One of these is a catalytically competent W species that cycles between the W(IV)/W(V)/W(VI) states at physiologically relevant potentials (<−300 mV) and gives rise to the “low-potential” W(V) resonance,  g  ∼ 1.99, 1.90, 1.86. This form of W is quantitatively and irreversibly converted into a distinct and inactive W(IV)/W(V) species by the addition of high concentrations of glycerol or ethylene glycol at 80 °C and is responsible for the “diol-inhibited” W(V) resonance,  g  ∼ 1.96, 1.94, 1.89. The other major form of W gives rise to a “high-potential” W(V) species,  g  ∼ 1.99, 1.96, 1.89, at nonphysiologically relevant potentials (>0 mV), as a result of a one-electron redox process that is tentatively attributed to ligand based oxidation of a W(VI) species. In addition, active samples of Pf AOR, in particular, can have up to 20% of the W as an inactive W(VI)/W(V) species, with a midpoint potential close to −450 mV, and is responsible for the “spin-coupled” W(V) resonance. This W(V) signal exhibits a broad complex resonance spanning 600 mT due to weak spin−spin interaction with the nearby  S  =  3 / 2  [4Fe−4S] +  cluster. Structures are proposed for each of the major W(V) species on the basis of EPR  g  values and  183 W  A  values as compared to other biological and synthetic W(V)/Mo(V) centers, VTMCD spectra, and the available X-ray crystallographic and XAS data for Pf AOR and the Mo-containing DMSO reductase from  Rhodobacter sphaeroides . Comparison with the limited spectroscopic data that are available for all known tungstoenzymes suggests two major classes of enzyme with distinct active site structures.