EXPRESSION AND CHARACTERIZATION OF HUMAN AND CHIMERIC HUMAN PARACOCCUS-DENITRIFICANS ELECTRON-TRANSFER FLAVOPROTEINS

Electron transfer flavoprotein (ETF) is a heterodimer that contains a single equivalent of FAD and accepts electrons from nine flavoprotein dehydrogenases in the mitochondrial matrix, Human ETF was expressed in Escherichia coli using the expression vector previously employed to e xpress Paracoccus denitrificans ETF (Bedzyk, L. A., Escudero, K. A., G ill, R. E., Griffin, K. J., and Frerman, F. E. (1993) J. Biol. Chem. 2 68, 20211-20217). cDNAs encoding the beta and alpha subunits of the hu man protein were inserted into the vector, mimicking the arrangement o f the P. denitrificans genes in which coding sequences are joined by o verlapping termination and initiation codons, A human ETF containing 3 0% P. denitrificans sequence at the amino terminus of the beta subunit was also expressed and purified, This chimeric ETF has 64% sequence i dentity with the human sequence in the substituted region, Kinetic con stants of medium chain and short chain acyl-CoA dehydrogenases for the chimeric ETFs were slightly changed from those of human ETF; but, the re are marked differences in the kinetic constants of sarcosine dehydr ogenase and electron transfer flavoprotein-ubiquinone oxidoreductase w ith the two ETFs, Absorption spectra of the three redox states of huma n, chimeric, and P. denitrificans ETF flavins are identical, However, the flavin circular dichroism spectra of the three ETFs are characteri stic for each species, The spectrum of the chimeric ETF has both human and P. denitrificans ETF features, The amplitude of the 436 nm band i s identical to that of the of the human ETF flavin, but the amplitude of the 375 nm band is identical to that of the P. denitrificans ETF fl avin, Thus, flavin in the chimeric ETF appears to be exposed to dipole s in the protein framework provided by human and bacterial sequences, These spectral data indicate that the flavin is located in the vicinit y of the amino-terminal region of the beta subunit, The kinetic data s uggest that the amino-terminal region of the beta subunit comprises pa rt of the docking site for some primary dehydrogenases and electron tr ansfer flavoprotein-ubiquinone oxidoreductase.