Fluorescence-lifetime measurements of FAD bound to lipoamide dehydrogenase from Azotohucter vineluizdii and Escherichiu coli were performed. It is shown from these results that the two FAD groups in the isolated dimeric enzyme, as well as in the enzyme in the intact complex of E. coli, are in non-equivalent surroundings. This contrasts with the near equivalence of the FAD groups of both the enzyme and complex isolated from A . vinelundii. Reduction of the complex with Mg", thiamine pyrophosphate and pyruvate or with NADH enables the attachment of a maleimide analogue specifically to the lipoyl moieties of the transacetylase(s). Spin label as detected by phase fluorimetry, it is shown that this fluorescent label is distributed in different micro-environments. The differences observed in energy transfer between fluorescent label, attached to the lipoyl moiety of the high-M, transacetylase, indicate different conformations of the complex from A . vinelandii. Upon introduction of the label after reduction with NADH a much larger energy transfer, thus a shorter distance, is observed between the label and FAD than when reduction is performed with Mg2+, thiamine pyrophosphate and pyruvate. A similar conformation dependence upon reduction is found for the pyruvate dehydrogenase complex from E. coli. It is thus proposed that the transacetylase of E. coli and the high-Mr transacetylase of A . vineluizdii are both non-symmetrically distributed within the complex.Earlier studies from this laboratory on the flavoprotein lipoamide dehydrogenase from pig heart [I ] showed that this dimeric enzyme containing identical peptide chains has different fluorescence lifetimes of its two FAD molecules. This indicates that the two prosthetic groups are in non-equivalent surroundings. The lower symmetry can arise from mutual interaction of the FAD molecules and is then of local nature. It is also possible that the symmetry of the enzyme peptide chains is affected by dimerization. This could be induced by the separation of lipoamide dehydrogenase from the complex. Therefore studies on the intact complex were performed. For this purpose the fluorescence lifetimes of the pyruvate dehydrogenase complexes from pig heart and Azotobucter vinelundii were measured at two temperatures. It appeared [2] that the lipoamide dehydrogenase from pig heart in the intact complex has also two non-equivalent FAD groups. This proves that the observed asymmetry is not induced during the purification procedure. The observation that the complex from A . vinelundii did not show this asymmetry of the FAD groups [2] indicated that the non-equivalence is perhaps typical for eucaryotic species.In the present studies properties of the lipoamide dehydrogenase in the pyruvate dehydrogenase complexes from A . vinelundii and Escherichiu coli were

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