Fluorescence Polarization and Energy‐Transfer Studies on the Pyruvate Dehydrogenase Complex of Escherichia coli

Abstract

We have attached eosin maleimide specifically to the lipoyl group of the pyruvate dehydrogenase complex isolated from Esclzerichia coli. Using this as the fluorescence acceptor and the intrinsic FAD of the lipoamide dehydrogenase subunit as the fluorescence donor, we confirmed previous measurements with other probes, in which it was suggested that the flavin moiety is at a substantial distance (over 4.5 nm) from the labeled lipoyl group. Since the lipoyl group must supply electrons to the FAD during the catalytic decarboxylation of pyruvate, we have investigated several potential mechanisms whereby this could happen. Movement within the complex, possibly triggered by the presence of substrate, seemed to be a strong possibility. Complex labeled with fluorophores on the accessible sulfhydryls, or on the lipoyl functions, did not give evidence of such triggering upon addition of substrate as judged by both static and dynamic fluorescence depolarization.The mobility of the subunits of labeled lipoamide dehydrogenase exceeded that expected for the total complex. Pyrene maleimide bound to the lipoyl functions also exhibited considerably faster rotations than the predicted one of the whole complex (z, > 3 ps). This suggests that a constant movement within the complex, coupled with the rotation of the lipoyl group, may bring the active sites of the complex transiently close enough together to interact on a time scale much faster than enzyme turnover. At the same time, the lipoyl group and the active sites of the complex can spend most of their time at points which are rather distant from each other.The pyruvate dehydrogenase complex from Eschericliia coli is a macromolecule with a molecular weight of at least 4.8 x lo6 [1,2] consisting of three enzymes (pyruvate dehydrogenase, lipoyl transacetylase and lipoamide dehydrogenase) which act in concert to cariry out the reactionThe pyruvate dehydrogenase and lipoamide dehydrogenase are noncovalently attached to the lipoyl transacetylase moiety which forms the core of the complex. The latter consists of 24 polypeptide subuni1.s each of which has two cofactor lipoic acid residues covalently bound by a lysyl amide bond. These lipolyl residues have been proposed to act as a 1.4-nmlong 'swinging arm' which passively transports acetyl enzyme^ The pyruvate dehydrogenase complex consists of pyruvate dehydrogenase, El (EC 1 2 4 l ) , lipoyl transacetylase, Ez (EC 2 3 1 12), and lipoamide dehydrogenase, E3 (EC 1 6 4 3 ) ~_ _ _ groups and electrons between the various subunit enzymes [ 3 ] . Recent evidence using fluorescence energy transfer techniques suggests, however, that the lipoyl groups when labeled with a fluorescent maleimide are at too great a distance (over 4.5 nm) from the FAD of the lipoamide dehydrogenase enzyme for the two to swing together in such a passive fashion [4-81. Similar measurements also place the lipoyl group at an equally long distance from the other active sites of the complex [9, lo].To date, all of the measurements of the distances between the flavin and ...

Rotational Diffusion of Eosin‐Labeled Pyruvate Dehydrogenase Complex of Escherichia coli

“…The results also indicate that eosin is folded back to the protein surface, contrary to that which has been observed with several spin and fluorescence probcs attached to the lipoyl groups of the core [4,6].…”

“…We investigated the fluorescence properties of this reporter group in relation to its function as acceptor of flavin excitation energy and with the possibility of monitoring conformational changes upon substrate addition [4]. Fluorescence experiments carried out on eosin bound to the lipoyl arm did not reveal motion within 10ns.…”

“…The complex isolated from Escherichia coli forms a highly symmetric arrangement of a core of 24 subunits of E2 surrounded by the other two subcnzymcs [3]. The lipoyl residues function as carrier of acetyl groups and electrons between the various enzyme subunits.In a previous publication we have described the specific attachrnent of eosin maleimide to the lipoyl groups of EZ [4]. We investigated the fluorescence properties of this reporter group in relation to its function as acceptor of flavin excitation energy and with the possibility of monitoring conformational changes upon substrate addition [4].…”

“…In a previous publication we have described the specific attachrnent of eosin maleimide to the lipoyl groups of EZ [4]. We investigated the fluorescence properties of this reporter group in relation to its function as acceptor of flavin excitation energy and with the possibility of monitoring conformational changes upon substrate addition [4].…”

Lipoamide dehydrogenase from Azotobacter vinelandii

“…Because this part of the enzyme is thought to be involved in substrate interaction [38], this tail could form an anchor to make contact with the transacetylase component of the multienzyme complexes. The binding of lipoamide dehydrogenase to this component is very flexible as indicated by fluorescence anisotropy measurements [4,40].…”