Evidence for the selective population of FeMo cofactor sites in MoFe protein and its molecular recognition by the Fe protein in transition state complex analogues of nitrogenase

We have collected synchrotron x-ray solution scattering data for the MoFe p rotein of KLebsiella pneumoniae nitrogenase and show that the molecular con formation of the protein that contains only one molybdenum per alpha (2)bet a (2) tetramer is different from that of the protein that has full occupanc y i.e. two molybdenums per molecule. This structural finding is consistent with the existence of MoFe protein molecules that contain only one FeMo cof actor site occupied and provides a rationale for the 50% loss of the specif ic activity of such preparations. A stable inactive transition state comple x has been shown to form in the presence of MgADP and AlF4-. Gel filtration chromatography data show that the MoFe protein lacking a full complement o f the cofactor forms initially a 1:1 complex before forming a low affinity 1:2 complex, A similar behavior is found for the MoFe protein with both cof actors occupied, but the high affinity 1:2 complex is formed at a lower rat io of Fe protein/MoFe protein. The 1:1 complex, MoFe protein-Fe protein . ( ADP . AlF4-)(2), formed with MoFe protein that lacks one of the cofactors, is stable. X-ray scattering studies of this complex have enabled us to obta in its low resolution structure at similar to 20-Angstrom resolution, which confirms the gel filtration finding that only one molecule of the Fe prote in binds the MoFe protein. By comparison with the low resolution structure of purified MoFe protein that contains only one molybdenum per tetramer, we deduce that the Fe protein interacts with the FeMo cofactor-binding alpha -subunit of the MoFe protein. This observation demonstrates that the confor mation of the alpha -subunit or the alpha beta subunit pair that lacks the FeMo cofactor is altered and that the change is recognized by the Fe protei n. The structure of the 1:1 complex reveals a similar change in the conform ation of the Fe protein as has been observed in the low resolution scatteri ng mask and the high resolution crystallographic study of the 1:2 complex w here both cofactors are occupied and with the Fe protein bound to both subu nits. This extensive conformational change observed for the Fe protein in t he complexes is, however, not observed when MgATP or MgADP binds to the iso lated Fe protein. Thus, the large scale conformational change of the Fe pro tein is associated with the complex formation of the two proteins.