The objectives of this group involve the extensive use of multinuclear magnetic resonance (NMR) techniques to elucidate the structure and/or function of a variety of biomolecules. When possible, specifically labeled (13C and 19F) amino acids are incorporated biosynthetically into strategic sites in an enzyme to enable one to focus on a particular aspect of a complex system. (gamma-13C)-histidine has been incorporated into E. coli alkaline phosphatase and the properties of the multiple metal binding sites have been characterized by 13C NMR studies on this labeled enzyme. From the 13C chemical shifts and the observation of 13C-113Cd spin coupling, 5 histidine residues are shown to function as metal ligands to the essential pair of metal ions which are located very close to one another in the subunit. These 13C NMR studies show the relative affinities of these site for Zn 2 ions to differ markedly for Cd2 ion. Substitution of 113 Cd 2 ion for the native Zn 2 ion allows the distribution of metals among all 6 metal sites/dimer to be directly monitored by 113 Cd NMR. These results provide a structural rationalization for the known 'half-sites" reactivity of all Me 2 ion enzymes and indicate that occupation of a pair of metal binding sites on a single subunit is a minimum requirement for substrate turnover. 113Cd NMR has also been used to study the properties of the metal binding sites in metallothionein, a small cysteine-rich metal binding protein, isolated from rabbit liver and mud crab following injection with enriched 113 CDCl2. The 113Cd spectrum exhibits extensive 113Cd-113Cd spin coupling which arises from adjacent 113Cd ions linked by bridging cysteine thiolate ligands. This data provided the first direct evidence that the metals (about 7 g-atoms metal/mole) exist in a polynuclear cluster arrangement. The number of metals in the clusters has also been found to vary for different metallothioneins.