Abstract
Electron-transferring flavoprotein (ETF) from the anaerobic bacterium Megasphaera elsdenii contains two FAD molecules. One FAD binds to the apoprotein relatively loosely so that it is released from the apoprotein in solution of high ionic strength e.g. 1 M KBr. The other FAD binds very tightly so that it is released only under denaturing conditions e.g. 6 M guanidine hydrochloride. In the absence of the loose FAD, the tight FAD shows normal flavin absorption spectrum, while the holoETF shows unusual spectrum: the valley at about 400 nm is very shallow. It seems likely that the loose FAD shows unusual flavin spectrum and the tight FAD shows normal spectrum in the holoETF. But there is another possibility that the loose FAD shows normal spectrum and the tight FAD shows unusual spectrum due to the environmental change caused by the binding of the loose FAD. To elucidate this subject, we prepared ETF which has 8-NH2-FAD at the tight site and no FAD at the loose site by denaturation-renaturation method. 8-NH2-FAD shows largely different spectrum from FAD, so that the spectral change due to the environmental change should be different from that of FAD. In fact, the spectral change upon binding of loose FAD to the ETF with tight 8-NH2-FAD was strictly the same as that to the ETF with tight FAD. This result indicates that the loose FAD not the tight FAD shows the unusual spectrum in the holoETF. [J Physiol Sci. 2008;58 Suppl:S198]
