Characterization of the γ protein and its involvement in the metallocluster assembly and maturation of dinitrogenase from Azotobacter vinelandii

Abstract

Dinitrogenase, the enzyme capable of catalyzing the reduction of N2, is a heterotetramer (α2β2) and contains the iron-molybdenum cofactor (FeMo-co) at the active site of the enzyme. Mutant strains unable to synthesize FeMo- co accumulate an apo form of dinitrogenase, which is enzymatically inactive but can be activated in vitro by the addition of purified FeMo-co. Apodinitrogenase from certain mutant strains of Azotobacter vinelandii has a subunit composition of α2β2γ2. The γ subunit has been implicated as necessary for the efficient activation of apodinitrogenase in vitro. Characterization of γ protein in crude extracts and partially pure fractions has suggested that it is a chaperone-insertase required by apodinitrogenase for the insertion of FeMo-co. There are three major forms of γ protein detectable by Western analysis of native gels. An apodinitrogenase-associated form is found in extracts of nifB or nifNE strains and dissociates from the apocomplex upon addition of purified FeMo-co. A second form of γ protein is unassociated with other proteins and exists as a homodimer. Both of these forms of γ protein can be converted to a third form by the addition of purified FeMo-co. This conversion requires the addition of active FeMo-co and correlates with the incorporation of iron into γ protein. Crude extracts that contain this form of γ protein are capable of donating FeMo-co to apodinitrogenase, thereby activating the apodinitrogenase. These data support a model in which γ protein is able to interact with both FeMo-co and apodinitrogenase, facilitate FeMo-co insertion into apodinitrogenase, and then dissociate from the activated dinitrogenase complex.