IDENTIFICATION OF A NITROGENASE PROTEIN-PROTEIN INTERACTION SITE DEFINED BY RESIDUES 59 THROUGH 67 WITHIN THE AZOTOBACTER-VINELANDII FE PROTEIN

During nitrogenase catalysis the Fe protein and the MoFe protein assoc iate and dissociate in a MgATP-dependent process involving electron tr ansfer from the Fe protein to the MoFe protein. A docking model, based primarily on the crystal structures of the separate components from A zotobacter vinelandii, was previously proposed in which the g-fold sym metric surface of the homodimeric Fe protein interacts with the expose d surface of a MoFe protein pseudosymmetric alpha beta-unit interface. In this model, a loop, which is included within residues 59 through 6 7 of the Fe protein primary sequence, is likely to interact with the M oFe protein during component protein docking. In the present study, ev idence supporting the component protein docking model was obtained by construction of an A. vinelandii strain that produces a hybrid Fe prot ein for which residues 59 through 67 have been replaced by the corresp onding residues from the Fe protein of Clostridium pasteurianum. Bioch emical analyses of the hybrid Fe protein revealed the following featur es when compared with the unaltered Fe protein. First, the hybrid Fe p rotein exhibited half the maximum specific activity of the normal Fe p rotein and was insensitive to inhibition by low levels of NaCl. Second , the hybrid Fe protein activity was hypersensitive to a molar excess of MoFe protein, which also resulted in the uncoupling of MgATP hydrol ysis from substrate reduction. Third, stopped-now spectrophotometry ex periments showed that during catalysis the hybrid Fe protein dissociat es from the MoFe protein at only half the normal rate of Fe protein-Mo Fe protein dissociation. Thus, the salient feature of the hybrid Fe pr otein is that it appears to form a relatively tighter complex with the MoFe protein. This property is in line with previous biochemical reco nstitution experiments where it was shown that a heterologous mixture of Fe protein hom C. pasteurianum and MoFe protein from A. vinelandii form a tight, inactive complex and supports the proposal that a region defined by residues 59 through 67 within the Fe protein is involved i n component protein interaction.