Evidence on intramolecular electron transfer in the MoFe protein of nitrogenase from Klebsiella pneumoniae from rapid-freeze electron-paramagnetic-resonance studies of its oxidation by ferricyanide.

A transient e.p.r. signal with g-values of 2.05, 1.95 and 1.81 was observed in rapid-freezing experiments when Kpl, the MoFe protein of nitrogenase from Klebsiella pneumoniae, was oxidized by ferricyanide or by some dyes. This e.p.r. signal was assigned to the 'P'-centres and, since such signals are characteristics of [4Fe-4S]1+ clusters, provides further evidence for the 'P'-centres being in the [4Fe-4S]0 oxidation level in the dithionite-reduced protein. When 4-10-fold excesses of ferricyanide were used as oxidants, the rate of disappearance of the transient e.p.r. signal was independent of the concentrations of ferricyanide, Kpl or ferrocyanide, i.e. its disappearance was by an intramolecular process. Under some circumstances the g = 3.7 e.p.r. signal from the FeMo-cofactors disappeared at a similar rate. It was concluded that, in these circumstances, the g = 3.7 e.p.r. signal disappears, owing to intramolecular electron transfer to the 'P'-centres in the [4Fe-4S]2+ (P2+) oxidation level, whereas the gav. = 1.933 transient e.p.r. signal from the P1+ centres disappears, owing to a change in its spin state from S = 1/2 to S = 5/2 the rate of this process being maximal when there are two P1+ centres in the half-molecule. The rate of the intramolecular decay of the e.p.r. signals, 4.1 +/- 0.8 s-1, is the same as the rate of enzyme turnover. It is suggested that both processes may be linked to the same conformational change, which triggers, or is triggered by, intramolecular electron transfer.