9317059 Roberts Dinitrogease is an important enzyme because of its role in the global nitrogen cycle. Because it is also one of the best understood complex metalloproteins, it is an excellent system for understanding how metal cluster are formed and inserted into apoproteins. This proposal addresses a central issue in that phenomenon, namely, the requirements and role of a protein (termed gamma) that associates with the apo-dinitrogenase (ApoI) and is necessary for the insertion of the iron-molybdenum cofactor (FeMoco) which serves as the active site of the enzyme. The results will have broad implications for protein folding and maturation, as well as for the mechanisms by which trace metals are efficiently utilized by cells. Active dinitrogenase is a A-2-B-2 complex comprised of the products of nifKD. The gene products of nifKD, N, E, and H are necessary for the synthesis of FeMoco and strains defective in any of these genes accumulate an active, apo-form of dinitrogenase. In the case of nifB, N, and E mutants, the apo-dinitrogenase is an A-2B-2 hexamer that is capable of being activated by purified FeMoco. In the case of nifH mutants, the apo-dinitrogenase exhibits as A-2B-2 structure and requires treatment with dinitrogenase reductase and MgATP before it can be activated by FeMoco. Because FeMoco is "buried" approximately 15A below the surface of active dinitrogenase, this insertion process must involve substantial alterations of Apol conformation. In Klebsiella pneumonia the gamma is the product of the nifY gene; in A. vinelandii, the identity of the gene encoding gamma is unknown, but it is not nifY. We have evidence that gamma is critical for insertion FeMoco in vitro and that it serves to stabilize Apol in vivo. We have also demonstrated that d dinitrogenase reductase is necessary to promote the association of gamma with Apol . Finally, we have partially purified an activity that stimulates the dissociation of gamma from the whole complex follo wing activation by FeMoco. We therefore have substantial body on information on the entire pathway by which FeMoco is added to ApoI and the aim of this proposal is to further define each of the steps in this pathway at a molecular level. The goals of this proposal are to (i) Define the mechanism by which gamma becomes associated with ApoI. This will involve purification of gamma from A. vinelandii, characterization of other proteins and small molecules that are involved in the association reaction, and identification of the gene that encodes gamma. (iii) Define the nature and requirements for the dissociation reaction. For this aim, we will purify the dissociation factor to homogeneity, characterize the requirements of the reaction, and identify the gene that encodes the protein. In each case, the proposed analysis specific will both address the specific aspects of dinitrogenase maturation and lay the groundwork for determining the larger cellular roles for the proteins involved. The information gained will provide a uniquely defined insight into metalloprotein maturation, but it will also addressed larger questions in the growing area of how certain proteins help others attain their final active conformation. %%% Dinitrogenase is an important enzyme because of its role in the global nitrogen cycle. It is also one of the best understood complex metalloproteeins and is an excellent system for understanding how metal clusters are formed and inserted into apo- proteins (proteins lacking their metal centers or cofactors). This proposal addresses a central issue in that phenomenon, namely, the requirements and role of a protein (termed gamma) that associates with the apo-dinitrogenase (Apol) and is necessary for the insertion of the iron-molybdenum cofactor (FeMoco) which serves as the active site of the enzyme. The results will have broad implications for protein folding and maturation, as well as for the mechanisms by which trace metals are efficiently util ized by cells. The information gained by this project will provide a uniquely defined insight into metalloprotein maturation, but will also address larger questions in the area of protein folding which is an active area of scientific investigation. In these studies the proteins and genes encoding the proteins will be isolated from bacterial models such as Klebsiella pneumoniae and Azotobacter vinelandii which are easily manipulated systems and part of the global biosphere involved in the nitrogen cycle. ***
