Multiple Acyl-CoA Dehydrogenase Deficiency with Variable Presentation Due to a Homozygous Mutation in a Bedouin Tribe

Multiple acyl-CoA dehydrogenase deficiency (MADD) is a fatty acid and amino acid oxidation defect caused by a deficiency of the electron-transfer flavoprotein (ETF) or the electron-transfer flavoprotein dehydrogenase (ETFDH). There are three phenotypes of the disease, two neonatal forms and one late-onset. Previous studies have suggested that there is a phenotype–genotype correlation. We report on six patients from a single Bedouin tribe, five of whom were sequenced and found to be homozygous to the same variant in the ETFDH gene, with variable severity and age of presentation. The variant, NM_004453.3 (ETFDH): c.524G>A, p.(R175H), was previously recognized as pathogenic, although it has not been reported in the literature in a homozygous state before. R175H is located near the FAD binding site, likely affecting the affinity of FAD for EFT:QO. The single homozygous ETFDH pathogenic variant was found to be causing MADD in this cohort with an unexpectedly variable severity of presentation. The difference in severity could partly be explained by early diagnosis via newborn screening and early treatment with the FAD precursor riboflavin, highlighting the importance of early detection by newborn screening.

Rapid kinetics reveal surprising flavin chemistry in the bifurcating electron transfer flavoprotein from Acidaminococcus fermentans.

Electron bifurcation uses free energy from exergonic redox reactions to power endergonic reactions. β-FAD of the electron transfer flavoprotein (EtfAB) from the anaerobic bacterium Acidaminococcus fermentans bifurcates the electrons of NADH, sending one to the low potential ferredoxin and the other to the high potential α-FAD semiquinone (α-FAD·-). The resultant α-FAD hydroquinone (α-FADH-) transfers one electron further to butyryl-CoA dehydrogenase (Bcd); two such transfers enable Bcd to reduce crotonyl-CoA to butyryl-CoA. To get insight into the mechanism of these intricate reactions, we constructed an artificial reaction only with EtfAB containing α-FAD or α-FAD·- to monitor formation of α-FAD·- or α-FADH-, respectively, using stopped flow kinetic measurements. In the presence of α-FAD, we observed that NADH transferred a hydride to β-FAD at a rate of 920 s-1, yielding the charge transfer complex NAD+:β-FADH- with an absorbance maximum at 650 nm. β-FADH- bifurcated one electron to α-FAD and the other electron to α-FAD of a second EtfAB molecule, forming two stable α-FAD·-. With α-FAD·-, the reduction of b-FAD with NADH was 1500-times slower. Reduction of β-FAD in the presence of α-FAD displayed a normal kinetic isotope effect (KIE) of 2.1, whereas the KIE was inverted in the presence of α-FAD·-. These data indicate that a nearby radical (14 Å apart) slows the rate of a hydride transfer and inverts the KIE. This unanticipated flavin chemistry is not restricted to Etf-Bcd but certainly occurs in other bifurcating Etfs found in anaerobic bacteria and archaea.

A family with riboflavin-reactive lipid deposition myopathy caused by a novel compound heterozygous mutation in the electron transfer flavoprotein dehydrogenase gene

We report a family with riboflavin-reactive multiple acyl-CoA dehydrogenase deficiency (RR-MADD) partially caused by a novel mutation in the electron transfer flavoprotein dehydrogenase gene (ETFDH). The RR-MADD family was identified by physical examination, electromyography, and muscle biopsy of the proband. Laboratory examination and electromyography suggested a muscle disease of the lipid storage myopathies. This was confirmed by a muscle biopsy that revealed lipid deposition in the muscle fibers. The proband’s sister previously had a similar disease, so the family underwent genetic testing. This revealed complex heterozygous ETFDH mutations c.389A > T (p. D130V) and c.1123C > A (p. P375T) in the proband and her sister, of which c.1123C > A (p. P375T) is a novel pathogenic mutation. The proband was treated with riboflavin and changes in physical symptoms and laboratory tests were evaluated before and after treatment. The discovery of a novel locus further expands the ETFDH mutation spectrum and suggests that genotyping is vital for early detection of RR-MADD as it can greatly improve the prognosis.

Spectroscopic evidence for direct flavin-flavin contact in a bifurcating electron transfer flavoprotein

A remarkable charge transfer (CT) band is described in the bifurcating electron transfer flavoprotein (Bf-ETF) from Rhodopseudomonas palustris (RpaETF). RpaETF contains two FADs that play contrasting roles in electron bifurcation. The Bf-FAD accepts electrons pairwise from NADH, directs one to a lower-reduction midpoint potential (E°) carrier, and the other to the higher-E° electron transfer FAD (ET-FAD). Previous work noted that a CT band at 726 nm formed when ET-FAD was reduced and Bf-FAD was oxidized, suggesting that both flavins participate. However, existing crystal structures place them too far apart to interact directly. We present biochemical experiments addressing this conundrum and elucidating the nature of this CT species. We observed that RpaETF missing either FAD lacked the 726 nm band. Site-directed mutagenesis near either FAD produced altered yields of the CT species, supporting involvement of both flavins. The residue substitutions did not alter the absorption maximum of the signal, ruling out contributions from residue orbitals. Instead, we propose that the residue identities modulate the population of a protein conformation that brings the ET-flavin and Bf-flavin into direct contact, explaining the 726 nm band based on a CT complex of reduced ET-FAD and oxidized Bf-FAD. This is corroborated by persistence of the 726 nm species during gentle protein denaturation and simple density functional theory calculations of flavin dimers. Although such a CT complex has been demonstrated for free flavins, this is the first observation of such, to our knowledge, in an enzyme. Thus, Bf-ETFs may optimize electron transfer efficiency by enabling direct flavin-flavin contact.