The NifZ accessory protein has an equivalent function in maturation of both nitrogenase MoFe protein P-clusters

The Mo-dependent nitrogenase comprises two interacting components called the Fe protein and the MoFe protein. The MoFe protein is an α₂β₂ heterotetramer that harbors two types of complex metalloclusters, both of which are necessary for N₂ reduction. One type is a 7Fe-9S-Mo-C-homocitrate species designated FeMo-cofactor, which provides the N₂-binding catalytic site, and the other is an 8Fe-7S species designated the P-cluster, involved in mediating intercomponent electron transfer to FeMo-cofactor. The MoFe protein's catalytic partner, Fe protein, is also required for both FeMo-cofactor formation and the conversion of an immature form of P-clusters to the mature species. This latter process involves several assembly factors, NafH, NifW, and NifZ, and precedes FeMo-cofactor insertion. Here, using various protein affinity-based purification methods as well as in vivo, EPR spectroscopy, and MALDI measurements, we show that several MoFe protein species accumulate in a NifZ-deficient background of the nitrogen-fixing microbe Azotobacter vinelandii These included fully active MoFe protein replete with FeMo-cofactor and mature P-cluster, inactive MoFe protein having no FeMo-cofactor and only immature P-cluster, and partially active MoFe protein having one αβ-unit with a FeMo-cofactor and mature P-cluster and the other αβ-unit with no FeMo-cofactor and immature P-cluster. Also, NifW could associate with MoFe protein having immature P-clusters and became dissociated upon P-cluster maturation. Furthermore, both P-clusters could mature in vitro without NifZ. These findings indicate that NifZ has an equivalent, although not essential, function in the maturation of both P-clusters contained within the MoFe protein.