9604446 Roberts Part 1 (Technical) Dinitrogenase is an important enzyme because of its role in the global nitrogen cycle, but it is also one of the best understood complex metalloproteins. This base of understanding makes it an excellent model system for understanding how metal clusters are formed and inserted into apo-proteins. This research addresses a central issue in that phenomenon, namely, the requirements and role of the ( protein that inserts the active site metal cluster into apodinitrogenase. The results will have broad implications for metal protein maturation, as well as for the mechanisms by which metals are efficiently utilized by cells. Active dinitrogenase is an (2 (2 complex with an Iron-Molybdenum cofactor (termed FeMo-co) at its active site. The PI identified a protein, termed ( , that is central to the maturation of dinitrogenase. ( is a homodimer that monomerizes upon the specific binding of FeMo-co. In the presence of dinitrogenase reductase and nucleotide, it donates that FeMo-co to ApoI, creating active dinitrogenasae. When FeMo-co is limiting, ( associates with ApoI, acting as a chaperone to stabilize ApoI in an altered conformation. ( is therefore a chaperone-insertase and this project will address a number of the critical features of that complex role. The monomer-dimer transition of ( upon binding FeMo-co and ('s chaperone role with ApoI suggest that these steps are supported by dramatic changes in protein structure and this research will seek to understand the nature and role of those structural changes. The goals of this proposal are the following: (i) Determination of the mechanism by which ( serves as a FeMo-co insertase. (ii) Development of a "structural" understanding of the role of ( in FeMo-co insertase and ApoI maturation. (iii) Identification of the requirements for dissociation of ( from ApoI following FeMo-co insertion. An activity that promotes the dissociation of ( from ApoI following FeMo-co had been detected and it will be pu rified and characterized. The information gained will provide a uniquely defined insight into metalloprotein maturation, particularly because many of the other relevant factors in the process have already been characterized. Because the structures of dinitrogenase and dinitrogenase reductase are known, the structure and properties of ( will serve to define the maturation process at the molecular level. It will also address the larger issue of how chaperones assist other proteins in attaining their final active conformation. Part 2 (Non-technical) Proteins that contain metal atoms perform many of the most critical functions in virtually all living organisms. It has been estimated that one-third of all enzymes require metals for function, and work over the past 20 years has provided a growing understanding of the role of metals in many of these enzymes. Surprisingly, however, very little is known about how metal atoms are actually placed within the appropriate enzymes. This process must involve a number of factors that have the ability to both identify and bind specific metal atoms and then to find the appropriate enzymes that should receive these specific metals. The PI identified one such protein, which inserts the critical metal complex into the active site of dinitrogenase, the enzyme responsible for all biological nitrogen fixation on earth. He will characterize the precise way in which this protein recognizes only one specific metal complex, how it protects that complex from degradation and how it inserts that complex into dinitrogenase. This process will certainly have similarities to the processing of other metal complexes and will therefore have broad biological implications.
