The enzyme transcarboxylase (TC) transfers a unit of CO2 from methylmalonyl-CoA to pyruvate to form oxalacetate. A key intermediate involves the 1.3S subunit of TC which binds CO2 on a biotin cofactor. The focus of this proposal is to delineate the structure, electron distribution and mechanism of action of the CO2-biotin moiety within the CO2-biotin-TC 1.3S complex. Raman and FT-IR difference spectroscopies will be used to delineate the structure and electronic distribution of the intermediate formed by carboxylated-biotin on the 1.3S subunit of the protein. Data interpretation will be aided by decarboxylation kinetics and vibrational spectra of model compounds resembling the CO2-biotin complex and by ab initio and vibrational calculations for these compounds. In order to set up the structure activity correlations for the CO2 biotin 1.3S subunit complex, site selected mutants of the protein which decarboxylate at different rates will be used.