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 pyruvate dehydrogenase (PDH) complex from Escherichia coli as studied in the scanning transmission electron microscope (STEM). The PDH complex is a multienzyme complex consisting of E₁, pyruvate dehydrogenase, E₂, dihydrolipoyl transacetylase, and E₃, dihydrolipoyl dehydrogenase. The core of the complex is the cubic 24-subunit E₂component, which contains the lipoyl moieties bonded to lipoyl-bearing domains. E₁and E₃are associated along the edges (E₁) and on the faces (E₃) of the core. The lipoyl moieties were reduced with NADH and alkylated with a p-maleimidobenzoyl undecagold cluster complex. The gold labels were found to be bound very nearly specifically by dihydrolipoyl transacetylase (E₂). Undecagold clusters were imaged directly by the STEM and also digitally mapped by radial mass analysis. The mass of the E₂E₃subcomplex is about half that of the PDH complex. The PDH complex and GC-PDH are both about 420 Å in diameter, as determined by radial mass analysis, and the E₂E₃subcomplex and GC-E₂E₃are 320 and 350 Å, respectively. The outer boundary of the E₂E₃subcomplex was clearly shown in STEM micrographs by the undecagold labels in GC-E₂E₃. Data obtained from radial mass analysis of GC-E₂E₃and the unlabeled E₂E₃subcomplex also showed that the size of the subcomplex is extended by the lipoyl-bearing domains surrounding the central E₂core. 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 many of the lipoyl groups in E₂E₃subcomplexes relax outward into space vacated by the removal of E₁during the preparation of the subcomplex 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 Å from the center of the complex, with the highest density at about 75 Å from the particle centers, near the interface between E₂and the associated components E₁and E₃.