PURIFICATION AND CHARACTERIZATION OF THE VNF-ENCODED APODINITROGENASEFROM AZOTOBACTER-VINELANDII

The unf-encoded apodinitrogenase (apodinitrogenase 2) has been purifie d from Azotobacter vinelandii strain CA117.30 (Delta nifKDB), and is a n alpha(2) beta(2) delta(2) hexamer. Apodinitrogenase 2 can be activat ed in vitro by the addition of the iron-vanadium cofactor (FeV-co) to form holodinitrogenase 2, which functions in C2H2, H+, and N-2 reducti on. Under certain conditions, the alpha(2) beta(2) delta(2) hexamer di ssociates to yield the free delta subunit (the VNFG protein) and a for m of apodinitrogenase 2 that exhibits no C2H2, H+, or N-2 reduction ac tivities in the in vitro FeV-co activation assay; however, these activ ities can be restored upon addition of VNFG to the FeV-co activation a ssay system. No other vnf-, nif-, or non-nif-encoded proteins were abl e to replace the function of VNFG in the in vitro processing of alpha( 2) beta(2) apodinitrogenase 2 (in the presence of FeV-co) to a form ca pable of substrate reduction, Apodinitrogenase 2 is also activable in vitro by the iron-molybdenum cofactor to form a hybrid enzyme with uni que properties, most notably the inability to reduce N-2 and insensiti vity to CO inhibition of C2H2 reduction.