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 (␣2␤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 ␣ 2 ␤ 2 ␥ 2 . The ␥ subunit has been implicated as necessary for the efficient activation of apodinitrogenase in vitro.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. There are three major forms of ␥ 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 ␥ protein is unassociated with other proteins and exists as a homodimer. Both of these forms of ␥ 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 ␥ protein. Crude extracts that contain this form of ␥ protein are capable of donating FeMo-co to apodinitrogenase, thereby activating the apodinitrogenase. These data support a model in which ␥ 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.Nitrogenase is comprised of two components: dinitrogenase (also known as component I or the MoFe protein) and dinitrogenase reductase (also known as component II, NifH, or the Fe protein). Nitrogenase catalyzes the ATP-and reductant-dependent reduction of N 2 and other substrates. Dinitrogenase is a 240-kDa ␣ 2 ␤ 2 tetramer encoded by nifKD (1). Dinitrogenase contains two types of metal centers: the P-cluster (8Fe-8S) (2, 3), which bridges the ␣ and ␤ subunits, and a unique ironmolybdenum cofactor (FeMo-co) 1 (4), which is buried within the ␣ subunit (3, 5) and is the site of substrate reduction (6). The dinitrogenase complex has two of each of these metal centers.Dinitrogenase reductase is a 60-kDa dimer encoded by nifH. It specifically reduces dinitrogenase, apparently transferring electrons to the P-cluster, which then channels them to FeMoco. In addition to this catalytic role, dinitrogenase reductase is also involved in the biosynthesis of FeMo-co and the maturation of dinitrogenase (reviewed in Ref.

The chaperone GroEL is required for the final assembly of the molybdenum-iron protein of nitrogenase

“…This result was not surprising because we had previously shown that a FeMoco-deficient form of the MoFe protein synthesized by a nifH deletion strain could be activated in cell-free extracts on addition of FeMoco, Fe protein, and MgATP but could not be activated by using the same additions once it was purified (22)(23)(24). We also knew from the literature that gamma and possibly other proteins and͞or factors were missing (24,25).…”

“…An E146D Fe protein was shown to be fully functional as an electron donor to the MoFe protein and in the initial biosynthesis of FeMoco. This Fe protein variant, however, was partially defective in the final assembly process such that the A. vinelandii strain expressing the E146D Fe protein accumulated both uninserted FeMoco [probably bound to gamma (25)] and a FeMoco-deficient form of the MoFe protein. Analysis of the purified MoFe protein from that strain showed that it was Ϸ55% active because of the fact that only Ϸ55% of its FeMoco sites were occupied (28).…”

“…It was previously established that the A. vinelandii strain expressing the E146D Fe protein accumulates uninserted FeMoco [presumably on gamma (25)] that can be captured in vitro by using a purified His-tag form of a FeMoco-deficient MoFe protein (28). Using the same method, the data in Table 1 establish that this uninserted FeMoco is still present in solutions of the partially purified E146D nifH MoFe protein, so that the final assembly assays described below did not require the addition of isolated FeMoco in NMF.…”

“…The FeMoco-deficient MoFe protein is then converted to a form with the FeMoco site accessible, and finally FeMoco is inserted. There may be a number of proteins and͞or factors involved in these final assembly steps, but to date it has been established only that the reaction requires the Fe protein, MgATP, and, in Azotobacter vinelandii, a FeMoco chaperone-insertase protein designated gamma (25). Here, we show that the final assembly of the MoFe protein also requires the molecular chaperone GroEL.…”

Characterization of VNFG, the δ Subunit of the vnf-Encoded Apodinitrogenase from Azotobacter vinelandii

“…The role of VNFG and ANFG might be analogous to the function of the ␥ protein in processing nif-encoded apodinitrogenase 1 (dinitrogenase 1 that does not contain FeMo-co) to dinitrogenase 1. The form of apodinitrogenase 1 that is activable by FeMo-co has an ␣ 2 ␤ 2 ␥ 2 composition (25); the ␥ protein, a nonnif-encoded gene product, has been shown to specifically asso-ciate with FeMo-co and perhaps function as a chaperone-insertase in the maturation of apodinitrogenase 1 to dinitrogenase 1 (26).…”