Conversion of a bifurcating ETF to a canonical ETF by site-directed mutagenesis.

In an effort to turn a bifurcating ETF (from Megasphaera elsdenii) into a canonical non-bifurcating ETF (from, e.g., Methylophilus methylotrophus), mutagenesis studies were performed to knock out the bf (bifurcating) FAD in Megasphaera elsdenii EtfAB. In order to determine the amino acid residues to mutate, the bf FAD binding site of Acidaminococcus fermentans (homolog to Megasphaera eldenii) was overlaid with the AMP binding site of Methylophilus methylotrophus. Two Megasphaera eldenii EtfAB variants were constructed, one with a single mutation (T132Y), and one with three (G130Q, T132Y, I234 M, also called 3X). These variants did not possess the bf FAD or reconstitute AMP in its place and could no longer be reduced by the median potential donor NADH. The oxidized spectra of the variants were very similar to the spectral contribution of the et (electron-transferring) FAD from the literature, indicating that there was no great difference in the immediate environment of the remaining et FAD. Both variants accumulated the same anionic semiquinone in the course of reductive titration by dithionite as did the wild-type EtfAB, but the variants exhibited an unusual, quite sharp absorption band at ∼730 nm that accumulated and disappeared over the course of titration. Addition of sulfite to the buffer removed this feature, which simplified the observed spectra while otherwise preserving the properties of the variants. In comparing the electron transfer of wild-type EtfAB and the variants, both the forward and reverse directions of electron transfer with bcd (Megasphaera eldenii ETF physiological partner) and the reduction by TMADH (Methylophilus methylotrophus physiological partner) were significantly slowed but still showed evidence of transfer. This work shows how drastically the activity of the et FAD is affected when the bf FAD is removed; however, it is not sufficient to convert a bifurcating ETF to a canonical ETF.