The functions of the flavin contact residues, αArg249 and βTyr16, in human electron transfer flavoprotein

Abstract

Arg249 in the large (α) subunit of human electron transfer flavoprotein (ETF) heterodimer is absolutely conserved throughout the ETF superfamily. The guanidinium group of αArg249 is within van der Waals contact distance and lies perpendicular to the xylene subnucleus of the flavin ring, near the region proposed to be involved in electron transfer with medium chain acyl-CoA dehydrogenase. The backbone amide hydrogen of αArg249 is within hydrogen bonding distance of the carbonyl oxygen at the flavin C(2). αArg249 may modulate the potentials of the two flavin redox couples by hydrogen bonding the carbonyl oxygen at C(2) and by providing delocalized positive charge to neutralize the anionic semiquinone and anionic hydroquinone of the flavin. The potentials of the oxidized/semiquinone and semiquinone/hydroquinone couples decrease in an αR249K mutant ETF generated by site directed mutagenesis and expression in Escherichia coli, without major alterations of the flavin environment as judged by spectral criteria. The steady state turnover of medium chain acyl-CoA dehydrogenase and glutaryl-CoA dehydrogenase decrease greater than 90% as a result of the αR249Ks mutation. In contrast, the steady state turnover of short chain acyl-CoA dehydrogenase was decreased about 38% when αR249K ETF was the electron acceptor. Stopped flow absorbance measurements of the oxidation of reduced medium chain acyl-CoA dehydrogenase/octenoyl-CoA product complex by wild type human ETF at 3°C are biphasic ( t 1/2=12 ms and 122 ms). The rate of oxidation of this reduced binary complex of the dehydrogenase by the αR249K mutant ETF is extremely slow and could not be reasonably estimated. αAsp253 is proposed to function with αArg249 in the electron transfer pathway from medium chain acyl-CoA dehydrogenase to ETF. The steady state kinetic constants of the dehydrogenase were not altered when ETF containing an αD253A mutant was the substrate. However, t 1/2 of the rapid phase of oxidation of the reduced medium chain acyl-CoA dehydrogenase/octenoyl-CoA charge transfer complex almost doubled. βTyr16 lies on a loop near the C(8) methyl group, and is also near the proposed site for interflavin electron transfer with medium chain acyl-CoA dehydrogenase. The tyrosine residue makes van der Waals contact with the C(8) methyl group of the flavin in human ETF and Paracoccus denitrificans ETF (as βTyr13) and lies at a 30°C angle with the plane of the flavin. Human βTyr16 was substituted with leucine and alanine residues to investigate the role of this residue in the modulation of the flavin redox potentials and in electron transfer to ETF. In βY16L ETF, the potentials of the flavin were slightly reduced, and steady state kinetic constants were modestly altered. Substitution of an alanine residue for βTyr16 yields an ETF with potentials very similar to the wild type but with steady state kinetic properties similar to βY16L ETF. It is unlikely that the β methyl group of the alanine residue interacts with the flavin C(8) methyl. Neither substitution of βTyr16 had a large effect on the fast phase of ETF reduction by medium chain acyl-CoA dehydrogenase.