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 ...

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INTRODUCTIONPyruvate dehydrogenase complexes from prokaryotes consist of three enzymes, TPP-containing pyruvate dehydrogenase (E,), lipoamide( Lips2)-containing lipoamide acetyltransferase ( E,), and FAD-containing dihydrolipoamide (HS-IipSH) dehydrogenase (E3). These enzymes act sequentially in the order given in the oxidative decarboxylation of pyruvate and the subsequent transfer of acetyl fragments to coenzyme A and reducing cquivalents to NAD ', according to the following reactions:Mg" pyruvate + TPP-E, ---[hydroxyethyl-TPPI-E, t CO,, (1)

The pyruvate dehydrogenase complex from Azotobucter vinelundii incubated with 0.05 -0.7 mM [2-14C]pyruvate, magnesium chloride and thiamine pyrophosphate under anaerobic conditions at 0 "C, incorporates four ['4C]acetyl groups per mole FAD which are bound to the highmolecular-weight lipoyl transacetylase. With 10 mM pyruvate, the low-molecular-weight lipoyl transacetylase is also labelled; to this enzyme 3-4 ['4C]acetyl groups are bound per mole FAD. This enzyme is not labelled when pyruvate is used in concentrations lower than 0.7 mM. The complex of A . vinehdii pre-labelled with non-radioactive N-ethylmaleimide did not incorporate label onto low-molecular-weight lipoyl transacetylase; a maximum of four acetyl groups are bound to the high-molecular weight lipoyl transacetylase.Maximum incorporation of four [14C]acetyl groups per mole FAD is found, within five seconds, on the lipoyl transacetylase component when the Escherichiu coli complex is labelled under anaerobic conditions, also in the presence of 10 mM pyruvate.In both complexes all incorporated acetyl groups are bound to sulfhydryl groups since they can be completely removed with hydroxylamine, by performic acid oxidation and by reaction with coenzyme A and arsenite. Under aerobic conditions a slow deacetylation is observed, which is blocked in the presence of N-ethylmaleimide.The multi-enzyme complex from A . vinelundii pre-labelled with non-radioactive N-ethylmaleimide and then labelled with N-ethyl [2,3-1"C]maleimide in the presence of pyruvate, magnesium chloride and thiamine pyrophosphate incorporates under anaerobic conditions at 0 "C, even at 10 mM pyruvate, a maximum of four N-ethyl['4C]maleimide groups per mole of FAD. The label is almost exclusively bound to the high-molecular-weight lipoyl transacetylase of the A . vinrfundii complex.The E. coli complex binds only 2-3 N-ethyl[14C]maleimide groups per mole of FAD under these conditions. Direct labelling and correction for labelling without pyruvate yields 4 -5 N-ethyl-[14C]maleimide per mole FAD.The low-molecular-weight lipoyl transacetylase can be removed by chromatography of the complex from A . vinelundii on a blue-dextran-Sepharose 4B column [De Abreu et ul. (1977) FEBS Lett. 82,89-921. The complex eluted from the column, consisting of the other three enzyme components, binds a maximum of four [14C]acetyl groups per mole of FAD, even with 10 mM pyruvate.It is concluded that the lipoyl/FAD ratio is four in the complexes as isolated by us from both sources. The interpretation of this ratio with respect to the stoichiometries of the different enzyme components will be discussed.The oxidative decarboxylation of pyruvate and the is catalyzed through the following sequence of reactions Pyruvate + thiamine-P2,--C02

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...