| Popis: |
Nitrogenase catalyzed substrate reduction requires electron transfer between two component proteins, the iron (Fe) protein and the molybdenum-iron (MoFe) protein in a reaction coupled to MgATP hydrolysis. Previous studies suggest that a single electron is transferred from the [4Fe-4S] cluster of the Fe protein to the [8Fe-7S] (P) cluster of the MoFe protein prior to being transferred to the site of substrate reduction in the MoFe protein at a unique [Mo-7Fe-9S]-homocitrate (M) cluster. While much information is available on the properties of the metal centers in the individual proteins, very little is known about the properties of these centers in the nitrogenase complex. Furthermore, the exact role of MgATP hydrolysis in electron transfer has not been established. In the present work, we have taken advantage of an altered Fe protein that binds tightly to the MoFe protein and catalyzes MgATP-independent primary electron transfer in order to address these two points. Previous mutagenesis experiments on the Fe protein have shown that deletion of Leu 127 results in an Fe protein that appears locked in a conformation similar to the wild-type Fe protein when MgATP is bound (Ryle, Seefeldt, 1996). Notably, both the wild-type Fe protein with MgATP bound and the L127A Fe protein have a midpoint potential (E m) of −420 mV. In addition, the L127Δ Fe protein was found to form a tight complex with the MoFe protein and could transfer a single electron to the MoFe protein (Lanzilotta et al, 1996). This work shows that the E m of the [4Fe-4S] cluster of the L127Δ Fe protein undergoes an additional shift from −420 to −620 mV when the L127Δ Fe protein binds to the MoFe protein. |
