Abstract
The electronic and redox properties of the iron and tungsten centers in the aldehyde ferredoxin oxidoreductases (AORs) from Pyrococcus furiosus (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 183W-enriched samples (183W, 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 183W 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.
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