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

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Abstract
The enzymatically reduced lipoyl residues of the transacetylase component of the pyruvate dehydrogenase complex from E. coli were labeled with eosin maleimide. [The pyruvate dehydrogenase complex consists of pyruvate dehydrogenase (EC 1.2.4.1), lipoyl transacetylase (EC 2.3.1.12) and lipoamide dehydrogenase, E3 (EC 1.6.4.3.).] Using eosin as triplet probe, triplet-triplet absorption dichroism measurements were performed to obtain rotational correlation times of the complex in the microsecond time domain. 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 the (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 on labeling.