MAPPING THE LIPOYL GROUPS OF THE PYRUVATE-DEHYDROGENASE COMPLEX BY USE OF GOLD CLUSTER LABELS AND SCANNING-TRANSMISSION ELECTRON-MICROSCOPY

This paper describes the organization of lipoyl moieties within the py ruvate dehydrogenase (PDH) complex from Escherichia coli as studied in the scanning transmission electron microscope (STEM). The PDH complex is a multienzyme complex consisting of E(1), pyruvate dehydrogenase, E(2), dihydrolipoyl transacetylase, and E(3), dihydrolipoyl dehydrogen ase. The core of the complex is the cubic 24-subunit E(2) component, w hich contains the lipoyl moieties bonded to lipoyl-bearing domains. E( 1) and E(3) are associated along the edges (E(1)) and on the faces (E( 3)) Of the core. The lipoyl moieties were reduced with NADH and alkyla ted with a p-maleimidobenzoyl undecagold cluster complex. The gold lab els were found to be bound very nearly specifically by dihydrolipoyl t ransacetylase (E(2)). Undecagold clusters were imaged directly by the STEM and also digitally mapped by radial mass analysis. The mass of th e E(2)E(3) subcomplex is about half that of the PDH complex. The PDH c omplex and GC-PDH are both about 420 Angstrom in diameter, as determin ed by radial mass analysis, and the E(2)E(3) subcomplex and GC-E(2)E(3 ) are 320 and 350 Angstrom, respectively. The outer boundary of the E( 2)E(3) subcomplex was clearly shown in STEM micrographs by the undecag old labels in GC-E(2)E(3). Data obtained from radial mass analysis of GC-E(2)E(3) and the unlabeled E(2)E(3) subcomplex also showed that the size of the subcomplex is extended by the lipoyl-bearing domains surr ounding the central E(2) core. The capabilities of lipoyl moieties to undergo translocation over long distances through structural mobility in the lipoyl-bearing domains was confirmed by the observation that ma ny of the lipoyl groups in E(2)E(3) subcomplexes relax outward into sp ace vacated by the removal of E(1) during the preparation of the subco mplex from PDH complex. Radial mass analysis of the PDH complex and GC -PDH indicates that lipoyl groups are distributed over a large region of the PDH complex, extending from the central core to 170-180 Angstro m from the center of the complex, with the highest density at about 75 Angstrom from the particle centers, near the interface between E(2) a nd the associated components E(1) and E(3).