Abstract
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.
Highlights
Characterization of ␥ 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
The results suggest a role for ␥ protein in which it interacts with both apodinitrogenase and FeMo-co; it holds the apodinitrogenase in a conformation that allows access to the FeMo-co ligand site, and it is capable of binding FeMo-co and subsequently donating it to apodinitrogenase
Fractions were analyzed for proteins by Western blots of anoxic native gels and of SDS-PAGE to identify the gamma-containing fractions and to ensure that ␥ protein was present in the expected form
Summary
Anoxic native gel electrophoresis was performed as described [8] except that the electrophoresis buffer was N2-sparged 65 mM Tris-glycine buffer (pH 8.6) containing 1.7 mM sodium dithionite. Activation of Apodinitrogenase with Purified FeMo-co—Buffers used throughout this work were sparged with N2 and contained 1.7 mM sodium dithionite. Fractions were analyzed for proteins by Western blots of anoxic native gels and of SDS-PAGE to identify the gamma-containing fractions and to ensure that ␥ protein was present in the expected form (described under “Results”). All fractions were assayed for their ability to provide FeMo-co to apodinitrogenase by adding the fractions to crude extract from A. vinelandii strain UW45 (nifB) or DJ1030 (nifBnifH). Purified fractions containing apodinitrogenase and unassociated ␥ protein were obtained as described [7] through the hydroxylapatite column step. SDS-PAGE followed by Western blotting with antibody to ␥ protein were performed on all the fractions
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