CHARACTERIZATION OF THE GAMMA-PROTEIN AND ITS INVOLVEMENT IN THE METALLOCLUSTER ASSEMBLY AND MATURATION OF DINITROGENASE FROM AZOTOBACTER-VINELANDII

Dinitrogenase, the enzyme capable of catalyzing the reduction of N2, i s a heterotetramer (alpha 2 beta 2) and contains the iron-molybdenum c ofactor (FeMo-co) at the active site of the enzyme. Mutant strains una ble to synthesize FeMo-co accumulate an apo form of dinitrogenase, whi ch is enzymatically inactive but can be activated in vitro by the addi tion of purified FeMo-co. Apodinitroge nase from certain mutant strain s of Azotobacter vinelandii has a subunit composition of alpha(2) beta (2) gamma(2). The gamma subunit has been implicated as necessary for t he efficient activation of apodinitrogenase in vitro. Characterization of gamma protein in crude extracts and partially pure fractions has s uggested that it is a chaperone-insertase required by apodinitrogenase for the insertion of FeMo-co. There are three major forms of gamma pr otein detectable by Western analysis of native gels. An apodinitrogena se-associated form is found in extracts of nifB or nifNE strains and d issociates from the apocomplex upon addition of purified FeMo-co. A se cond form of gamma protein is unassociated with other proteins and exi sts as a homodimer. Both of these forms of gamma protein can be conver ted to a third form by the addition of purified FeMo-co. This conversi on requires the addition of active FeMo-co and correlates with the inc orporation of iron into gamma protein. Crude extracts that contain thi s form of gamma protein are capable of donating FeMo-co to apodinitrog enase, thereby activating the apodinitrogenase. These data support a m odel in which gamma 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.