Evidence for Coupled Electron and Proton Transfer in the [8Fe-7S] Cluster of Nitrogenase

Abstract

Substrate reduction by nitrogenase requires electron transfer from a [4Fe-4S] cluster in the iron (Fe) protein component to an FeMo cofactor in the molybdenum−iron (MoFe) protein component in a reaction that is coupled to MgATP hydrolysis and component protein association and dissociation. An [8Fe-7S] (or P-) cluster in the MoFe protein has been proposed as an intermediate electron-transfer site, although how this cluster functions in electron-transfer remains unclear. In the present work, it is demonstrated that one redox couple of the P-cluster (P^2+/1+) undergoes coupled electron and proton transfer, whereas a more reduced couple (P^1+/N) does not involve a coupled proton transfer. Redox titrations of the MoFe protein P-cluster were performed, and the midpoint potential of the P^2+/1+ couple (E_m2) was found to be pH dependent, ranging from −224 mV at pH 6.0 to −348 mV at pH 8.5. A plot of E_m2 versus the pH for this redox couple was linear and revealed a change of −53 mV/pH unit, indicating a single protonation event associated with reduction. From this plot, it was concluded that p is 8.5 in a proton-modified Nernst equation. In contrast, the midpoint potential for the P^1+/N couple (E_m1) was found to be −290 mV and was invariant over the pH range 6.0−8.5. These results indicate that the protonated species does not influence either the P¹⁺ or the P^N oxidation states. In addition, at physiological pH values, electron transfer is coupled to proton transfer for the P^2+/1+ couple. The P-clusters are unique among [Fe-S] clusters in that they appear to be ligated to the protein through a serinate-γO ligand (βSer188) and a peptide bond amide-N ligand (αCys88), in addition to cysteinate-S ligands. Elimination of the serinate ligand by replacement with a glycine was found to shift the E_m values for both P-cluster couples by greater than +60 mV, however the pH dependence of E_m2 was unchanged. These results rule out Ser188 as the protonated ligand responsible for the pH dependence of E_m2. The implications of these results in understanding the nitrogenase electron-transfer mechanism are discussed.