Redox chemistry of tungsten and iron-sulfur prosthetic groups in Pyrococcus furiosus formaldehyde ferredoxin oxidoreductase.
Formaldehyde oxidoreductase (FOR) is one of the tungstopterin iron-sulfur enzymes of the five-membered family of aldehyde oxidoreductases in the hyperthermophilic archaeon Pyrococcus furiosus. In dye-mediated equilibrium redox titrations, the tungsten in active P. furiosus FOR is a two-electron acceptor, W(VI/IV). The intermediate, paramagnetic W(V) state can be trapped only by reduction with substrate, with consecutive one-electron intraprotein electron transfer to the single [4Fe-4S](2+;+) cluster and partial comproportionation of the tungsten over W(IV, V, VI); this is a stable state in the absence of an external electron acceptor. Electron paramagnetic resonance (EPR) spectroscopy reveals a single "low-potential" W(V) spectrum with gxyz values 1.847, 1.898, and 1.972, and a [4Fe-4S]+ cubane in a spin mixture of S = 1/2 (10%) and S = 3/2 (90%) of intermediate rhombicity (E/D = 0.21, greal = 1.91). The development of this intermediate in vitro is slow even at elevated temperature and with a nominal 50:1 excess of substrate over enzyme presumably owing to the very unfavorable hydration equilibrium of the formaldehyde/methylene glycol couple with KD approximately 10(3). Rapid intermediate formation of enzyme at concentrations suitable for EPR spectroscopy (200 microM) is only obtained with extremely high nominal substrate concentration (1 M formaldehyde) and is followed by a slower phase of denaturation. The premise that the free formaldehyde, and not the methylene glycol, is the enzyme's substrate implies that KM for formaldehyde is 3 orders of magnitude less that the previously reported value.