m d lnatitut Curi.:. Unitc 219 ( I n h u t National di. la SmtC ;I d< la Ri~cherchi. Mkdicale). Orsay (R,>c?iv:d Jun; 1. 1981) The enzymatically reduced lipoyl residues of the transacetylase component of the pyruvate dehydrogenase complex from Eschericlzia coli were labeled with eosin maleimide. Using eosin as triplet probe, triplet-triplet absorption dichroism measurements were pcrformed to obtain rotational correlation times of the complex in the microsecond time domain. It was found that the hydrodynamic properties determined from the correlation times are in very good agreement with those obtained with other methods of different origin. The results can be fully explained by eosin molecules rotating with the whole complex, which consists of a mixture of heavy (60 S) and light (20 S) particles. Since no independent mobility could be detected it is suggested that thc (charged) chromophoric group is folded against the protein surface. Labeling with excess eosin maleimide tends to destabilize the complex. since the longer correlation time (60 S) decreases and the contribution of the shorter correlation time (20 S) becomes more significant upon labeling.The pyruvate dehydrogenasc multienzyme complex consists in its most elementary form of three subenzymes: pyruvatc dehydrogenase (El), dihydrolipoyl transacetylase (Ez) and dihydrolipoyl dehydrogenase (E3). The three enzymes catalyze the decarboxylation of pyruvate and formation of acetyl-CoA through a reaciion sequence, which is extensively described in the litcraturc [ I , 21. 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]. Fluorescence experiments carried out on eosin bound to the lipoyl arm did not reveal motion within 10ns. However because of the short lifetime of the fluorescence it was not possible to detect slower structural fluctuations as observed by using pyrene malcimide as conjugated dye.Eosin derivatives have the property that they can be efficiently excited into the much longer living triplet state 151, rendering slower motions to be investigated. We have measured triplet absorption dichroism of eosin-conjugated pyruvate dehydrogenase complex. The resulting data can be satisfkctorily interpreted with the supposition that the dye follows the rotation of the whole complex consisting of differently sedimenting particles (60 S and 20 S). No effect of substrate addition could be found, which implies that the cosin molecules did not sense triggered conformational Ei77l?mcx Thi. pyruvatc dchydrogenase comp...
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