Pyrococcus furiosus glyceraldehyde 3‐phosphate oxidoreductase has comparable W6+/5+ and W5+/4+ reduction potentials and unusual [4Fe‐4S] EPR properties

Pyrococcus furiosus glyceraldehyde 3‐phosphate oxidoreductase has been characterized using EPR‐monitored redox titrations. Two different W signals were found. W1 5+ is an intermediate species in the catalytic cycle, with the midpoint potentials E m(W6+/5+)=−507 mV and E m(W5+/4+)=−491 mV. W2 5+ represents an inactivated species with E m(W6+/5+)=−329 mV. The cubane cluster exhibits both S=3/2 and S=1/2 signals with the same midpoint potential: E m([4Fe‐4S]2+/1+)=−335 mV. The S=1/2 EPR signal is unusual with all g values below 2.0. The titration results combined with catalytic voltammetry data are consistent with electron transfer from glyceraldehyde 3‐phosphate first to the tungsten center, then to the cubane cluster and finally to the ferredoxin.