AN ALL-FERROUS STATE OF THE FE PROTEIN OF NITROGENASE - INTERACTION WITH NUCLEOTIDES AND ELECTRON-TRANSFER TO THE MOFE PROTEIN

The MoFe protein of nitrogenase catalyzes the six-electron reduction o f dinitrogen to ammonia. It has long been believed that this protein r eceives the multiple electrons it requires one at a time, from the [4F e-4S](2+/+) couple of the Fe protein. Recently an all-ferrous [4Fe4S]( 0) state of the Fe protein was demonstrated suggesting instead a serie s of two electron steps involving the [4Fe-4S](2+/0) couple. We have e xamined the interactions of the [4Fe-4S](0) Fe protein with nucleotide s and its ability to transfer electrons to the MoFe protein. The [4Fe4 5](0) Fe protein binds both MgATP and MgADP and undergoes the MgATP in duced conformational change and then binds properly to the MoFe protei n, as evidenced by the fact that the behavior of the 0 and +1 oxidatio n states in the chelation and chelation protection assays are indistin guishable. Nucleotide binding does not effect the distinctive UV/Vis, CD, or Mossbauer spectra exhibited by the [4Fe-4S](0) Fe protein; howe ver, because the intensity of the g = 16.4 EPR signal of the [4Fe-4S]( 0) Fe protein is extremely sensitive to minor variations of the rhombi city parameter E/D, the EPR signal is sensitive to the binding of nucl eotides. A 50:50 mixture of [4Fe-4S](2+) and [4Fe-4S](0) Fe protein re sults in electron self-exchange and 100% production of [4Fe-4S](2+) Fe protein, demonstrating that the +1/0 couple is fully reversible. MgAT P is absolutely required for electron transfer from the [4Fe-4S](0) Fe protein to the reduced state of the MoFe protein. In that reaction bo th electrons are transferred and are used to reduce substrate.