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, is a heterotetramer (alpha 2 beta 2) 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 alpha 2 beta 2 gamma 2. The gamma subunit has been implicated as necessary for the efficient activation of apodinitrogenase in vitro. Characterization of gamma 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. These are three major forms of gamma 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 gamma protein is unassociated with other proteins and exists as a homodimer. Both of these forms of gamma 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 gamma protein. Crude extracts that contain this form of gamma protein are capable of donating FeMo-co to apodinitrogenase, thereby activating the apodinitrogenase. These data support a model 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.
Homer
M J
MJ
Department of Bacteriology, University of Wisconsin, Madison 53706, USA.
Dean
D R
DR
Roberts
G P
GP
D016948:Azotobacter vinelandii / Q000201:enzymology*; D007501:Iron / Q000737:chemistry*; D008982:Molybdenum / Q000737:chemistry*; D009591:Nitrogenase / Q000737:chemistry* / Q000235:genetics / Q000502:physiology
D008982:Molybdenum; D007501:Iron; D009591:Nitrogenase