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

• Published in: The Journal of biological chemistry 2001-03, Vol.276 (9), p.6582-6590
• Main Authors: Grossmann, J. Günter, Hasnain, S. Samar, Yousafzai, Faridoon K., Eady, Robert R.
• Format: Article
• Language: English
• Subjects: Adenosine Diphosphate - metabolism; Adenosine Triphosphate - metabolism; Crystallography; Klebsiella pneumoniae; MoFe protein; Molybdoferredoxin - chemistry; Oxidoreductases - chemistry; Protein Conformation; Protein Subunits
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M005350200

We have collected synchrotron x-ray solution scattering data for the MoFe protein of Klebsiella pneumoniae nitrogenase and show that the molecular conformation of the protein that contains only one molybdenum per α2β2 tetramer is different from that of the protein that has full occupancy i.e. two molybdenums per molecule. This structural finding is consistent with the existence of MoFe protein molecules that contain only one FeMo cofactor site occupied and provides a rationale for the 50% loss of the specific activity of such preparations. A stable inactive transition state complex 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 of 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 cofactors occupied, but the high affinity 1:2 complex is formed at a lower ratio 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 obtain its low resolution structure at ∼20-Å resolution, which confirms the gel filtration finding that only one molecule of the Fe protein 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 α-subunit of the MoFe protein. This observation demonstrates that the conformation of the α-subunit or the αβ subunit pair that lacks the FeMo cofactor is altered and that the change is recognized by the Fe protein. The structure of the 1:1 complex reveals a similar change in the conformation of the Fe protein as has been observed in the low resolution scattering mask and the high resolution crystallographic study of the 1:2 complex where both cofactors are occupied and with the Fe protein bound to both subunits. This extensive conformational change observed for the Fe protein in the complexes is, however, not observed when MgATP or MgADP binds to the isolated Fe protein. Thus, the large scale conformational change of the Fe protein is associated with the complex formation of the two proteins.