Evidence for coupled electron and proton transfer in the [8Fe-7S] cluster of nitrogenase.
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 (P2+/1+) undergoes coupled electron and proton transfer, whereas a more reduced couple (P1+/N) does not involve a coupled proton transfer. Redox titrations of the MoFe protein P-cluster were performed, and the midpoint potential of the P2+/1+ couple (Em2) was found to be pH dependent, ranging from -224 mV at pH 6.0 to -348 mV at pH 8.5. A plot of Em2 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 <6.0 and p is >8.5 in a proton-modified Nernst equation. In contrast, the midpoint potential for the P1+/N couple (Em1) 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 PN oxidation states. In addition, at physiological pH values, electron transfer is coupled to proton transfer for the P2+/1+ couple. The P-clusters are unique among [Fe-S] clusters in that they appear to be ligated to the protein through a serinate-gammaO ligand (betaSer188) and a peptide bond amide-N ligand (alphaCys88), in addition to cysteinate-S ligands. Elimination of the serinate ligand by replacement with a glycine was found to shift the Em values for both P-cluster couples by greater than +60 mV, however the pH dependence of Em2 was unchanged. These results rule out Ser188 as the protonated ligand responsible for the pH dependence of Em2. The implications of these results in understanding the nitrogenase electron-transfer mechanism are discussed.
Lanzilotta
W N
WN
Department of Chemistry and Biochemistry, Utah State University, Logan 84322, USA.
Christiansen
J
J
Dean
D R
DR
Seefeldt
L C
LC
D019943:Amino Acid Substitution; D016948:Azotobacter vinelandii; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D005998:Glycine / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D046911:Macromolecular Substances; D008983:Molybdoferredoxin / Q000737:chemistry / Q000378:metabolism; D009591:Nitrogenase / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D011522:Protons; D012694:Serine / Q000378:metabolism
D046911:Macromolecular Substances; D008983:Molybdoferredoxin; D011522:Protons; D012694:Serine; D007501:Iron; D009591:Nitrogenase; D005998:Glycine