Cryoelectron Microscopy of Frozen-Hydrated Pyruvate Dehydrogenase Complexes

Abstract

Pyruvate dehydrogenase complexes (PDC’s) from both prokaryotes and eukaryotes exist as large multisubunit assemblies in which one of the component enzymes, dihydrolipoyl transacetylase (E2), forms a structural core that possesses multiple binding sites on its surface for the pyruvate dehydrogenase (E1) and dihydrolipoyl dehydrogenase (E3) components. We are investigating the structure of PDC’s fromE. coliand bovine heart by cryoelectron microscopy of frozen-hydrated native complexes and various types of subcomplexes prepared in vitro. Fields of frozen-hydrated PDC fromE. coliand bovine heart are shown in Figures 1 and 2 respectively. In many images of theE. coliPDC (M.W. ∽ 5 × 106) the E2 core is visible as a dense square or rectangular mass at the center of the images (see inset in Fig. 1). The E1 and E3 components surround the E2 core, and do not appear to form a symmetrical pattern. Furthermore, there are often apparent gaps of up to several nanometers separating the bound subunits from the surface of the core. These observations suggest that the E1 and/or E3 are linked to the E2 core by flexible polypeptide ‘tethers’ which could be important in the mechanism of active site coupling. Similar observations apply to the images of the larger PDC from bovine heart (M.W. ∽ 9 × 106).Selected images of frozen-hydrated E2 cores isolated fromE. coliand heart PDC are shown in Figures 3 and 4. TheE. coliE2 core consists of 24 identical polypeptides arranged with octahedral symmetry, whereas the larger heart core complex consists of about 60 identical polypeptides apparently arranged with icosahedral symmetry (another component, present in a few copies and referred to as component X, also appears to be integrated into the core complex). Each E2 polypeptide possesses specialized domains (three inE. coli. two in heart) which contain the lipoic acid cofactor and are thought to be involved in coordinating the sequence of reactions catalyzed. TheE. coliE2 cores often appear surrounded by a faint “fuzz” extending out from the surface by about 10 nm which we attribute to the lipoyl-bearing domains. Independent biochemical and biophysical observations are consistent with this interpretation. The larger heart E2 cores exhibit three main image types that probably correspond to views approximately along 5-fold, 3-fold and 2-fold symmetry axes. The fuzzy material observed surrounding theE. colicores is not readily visible in the heart E2, probably because there are fewer lipoyl-bearing domains and because they occupy a much larger volume than inE. coli. Methodologies for characterizing the spatial distribution of the lipoyl-bearing domains by EM, such as labeling them with monoclonal antibodies, are being pursued.