Expression and characterization of human and chimeric human-Paracoccus denitrificans electron transfer flavoproteins.

摘要: Electron transfer flavoprotein (ETF) is a heterodimer that contains single equivalent of FAD and accepts electrons from nine dehydrogenases in the mitochondrial matrix. Human ETF was expressed Escherichia coli using expression vector previously employed to express Paracoccus denitrificans (Bedzyk, L. A., Escudero, K. W., Gill, R. E., Griffin, J., Frerman, F. E. (1993) J. Biol. Chem. 268, 20211-20217). cDNAs encoding beta alpha subunits human protein were inserted into vector, mimicking arrangement P. genes which coding sequences are joined by overlapping termination initiation codons. A containing 30% sequence at amino terminus subunit also purified. This chimeric has 64% identity with substituted region. Kinetic constants medium chain short acyl-CoA for ETFs slightly changed those ETF; but, there marked differences kinetic sarcosine dehydrogenase electron flavoprotein-ubiquinone oxidoreductase two ETFs. Absorption spectra three redox states human, chimeric, flavins identical. However, flavin circular dichroism characteristic each species. The spectrum both features. amplitude 436 nm band identical flavin, but 375 flavin. Thus, appears be exposed dipoles framework provided bacterial sequences. These spectral data indicate located vicinity amino-terminal region subunit. suggest comprises part docking site some primary oxidoreductase.