Fluorescence Energy‐Transfer Studies on the Pyruvate Dehydrogenase Complex Isolated from Azotobacter vinelandii

Abstract

Fluorescence energy transfer has been employed to estimate the minimum distance between each of the active sites of the 4 component enzymes of the pyruvate dehydrogenase multienzyme complex from Azotobacter vinelandii. No energy transfer was seen between thiochrome diphosphate, bound to the pyruvate decarboxylase active site, and the FAD of the lipoamide dehydrogenase active site. Likewise, several fluorescent sulfhydryl labels, which were specifically bound to the lipoyl moiety of lipoyl transacetylase, showed no energy transfer to either the flavin or thiochrome diphosphate. These observations suggest that all the active centers of the complex are quite far apart ( 2 40 nm), at least during some stages of catalysis. These results do not preclude the possibility that the distances change during catalysis.Several of the fluorescent probes used possessed multiple fluorescent lifetimes, as shown by determination of lifetime averages by both phase and modulation measurements on a phase fluorimeter. These lifetimes are shown to result from multiple factors, not necessarily related to multiple protein conformations.The active sites of the four enzyme components of the pyruvate dehydrogenase complex are either fluorescent per se or can be specifically labeled with fluorescent probes. Using energy transfer measurements from fluorescence donor-acceptor pairs, minimum distances between these components can be calculated. Recently, Moe and Hammes [l] have shown, using the enzyme complex isolated from Escherichia coli, that the thiamine pyrophosphate binding site of the pyruvate decarboxylase portion of the complex is at least 4 nm from the flavin component of the lipoamide dehydrogenase moiety. Shepherd et al. [2] have likewise shown that the flavin moiety of lipoamide dehydrogenase is at least 5 nm from the CoA binding site of the lipoyl transacetylase portion of the E. coli pyruvate dehydrogenase complex.We demonstrate here that the active sites of all four component enzymes of the pyruvate dehydrogenase complex isolated from Azotobacter vinelandii are also at least 4 nm apart, as shown by fluorescenceenergy transfer. Of the various models of enzyme action that could be reconciled with these data, the most attractive, we believe, is that which involves conformational changes, which alter the distances between the various enzyme components during catalysis. MATERIALS AND METHODSThe pyruvate dehydrogenase complex from A . vinelandii was isolated by the method of Bresters et al.[3]. The specific activity of the complex was 8 -12 pmol NADH oxidized min Fluorescence lifetimes were measured on a phase fluorimeter, described elsewhere [4], and operated with a 60-MHz modulated light beam. The excited light was from a mercury lamp, connected to a Jarrel Ash 0.25-m grating monochromator in order to isolate the characteristic mercury emission lines. The emitted light was filtered through a series of broad band and/or narrow band filters, as needed, to eliminate scattered light or light from other fluorophores that were present. mg...