Purification and characterization of the vnf-encoded apodinitrogenase from Azotobacter vinelandii

The unf-encoded apodinitrogenase (apodinitrogenase 2) has been purified from Azotobacter vinelandii strain CA117.30 (Delta nifKDB), and is an alpha(2) beta(2) delta(2) hexamer. Apodinitrogenase 2 can be activated in vitro by the addition of the iron-vanadium cofactor (FeV-co) to form holodinitrogenase 2, which functions in C2H2, H+, and N-2 reduction. Under certain conditions, the alpha(2) beta(2) delta(2) hexamer dissociates to yield the free delta subunit (the VNFG protein) and a form of apodinitrogenase 2 that exhibits no C2H2, H+, or N-2 reduction activities in the in vitro FeV-co activation assay; however, these activities can be restored upon addition of VNFG to the FeV-co activation assay system. No other vnf-, nif-, or non-nif-encoded proteins were able 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 capable of substrate reduction, Apodinitrogenase 2 is also activable in vitro by the iron-molybdenum cofactor to form a hybrid enzyme with unique properties, most notably the inability to reduce N-2 and insensitivity to CO inhibition of C2H2 reduction.