GENETIC-ANALYSIS ON THE NIFW BY UTILIZING THE YEAST 2-HYBRID SYSTEM REVEALED THAT THE NIFW OF AZOTOBACTER-VINELANDII INTERACTS WITH THE NIFZ TO FORM HIGHER-ORDER COMPLEXES

Nitrogenase is a complex metalloenzyme composed of two separately puri fied proteins designated the Fe-protein and the MoFe-protein. Apart fr om these two proteins, a number of accessory proteins are essential fo r the maturation and assembly of nitrogenase. Even though experimental evidence suggests that these accessory proteins are required for nitr ogenase activity, the exact roles played by many of these proteins in the functions of nitrogenase are unclear. Our studies were directed to understand the role of two nif accessory proteins, the NifW and the N ifZ in the biological nitrogen fixation. To accomplish this, we have u tilized a genetic method, the Yeast based Two-Hybrid protein-protein i nteraction assay. This analysis showed that the NifW could interact wi th itself to make a multimeric complex. In contrast, the NifZ could no t interact with itself. However, the NifZ could interact with the NifW . Previously it was shown that mutating either the NifW or the NifZ ha ve similar effects on the activity of nitrogenase. This observation in dicated that both these proteins may exert their regulation on the nit rogenase by a common pathway. Furthermore, it was suggested that the N ifW plays a role in the oxygen-protection of the MoFe-protein by direc t physical interaction. Our observation that the NifW can interact wit h itself as well as with the NifZ, suggests that the NifW and the NifZ may form a higher order complex and such a complex may be needed to e xert the effects of the NifW or the NifZ on the nitrogenase activity. (C) 1998 Academic Press.