The crystal structure of electron transfer flavoprotein (ETF) from Paracoccus denitrificans was determined and refined to an R-factor of 19.3% at 2.6 A resolution. The overall fold is identical to that of the human enzyme, with the exception of a single loop region. Like the human structure, the structure of the P. denitrificans ETF is comprised of three distinct domains, two contributed by the alpha-subunit and the third from the beta-subunit. Close analysis of the structure reveals that the loop containing betaI63 is in part responsible for conferring the high specificity of AMP binding by the ETF protein. Using the sequence and structures of the human and P. denitrificans enzymes as models, a detailed sequence alignment has been constructed for several members of the ETF family, including sequences derived for the putative FixA and FixB proteins. From this alignment, it is evident that in all members of the ETF family the residues located in the immediate vicinity of the FAD cofactor are identical, with the exception of the substitution of serine and leucine residues in the W3A1 ETF protein for the human residues alphaT266 and betaY16, respectively. Mapping of ionic differences between the human and P. denitrificans ETF onto the structure identifies a surface that is electrostatically very similar between the two proteins, thus supporting a previous docking model between human ETF and pig medium-chain acyl-CoA dehydrogenase (MCAD). Analysis of the ionic strength dependence of the electron transfer reaction between either human or P. denitrificans ETF and MCAD demonstrates that the human ETF functions optimally at low ( approximately 10 mequiv) ionic strength, while P. denitrificans ETF is a better electron acceptor at higher (>75 mequiv) ionic strength. This suggests that the electrostatic surface potential of the two proteins is very different and is consistent with the difference in isoelectric points between the proteins. Analysis of the electrostatic potentials of the human and P. denitrificans ETFs reveals that the P. denitrificans ETF is more negatively charged. This excess negative charge may contribute to the difference in redox potentials between the two ETF flavoproteins and suggests an explanation for the opposing ionic strength dependencies for the reaction of MCAD with the two ETFs. Furthermore, by analysis of a model of the previously described human-P. denitrificans chimeric ETF protein, it is possible to identify one region of ETF that participates in docking with ETF-ubiquinone oxidoreductase, the physiological electron acceptor for ETF.

Defects in electron transfer flavoprotein (ETF) or its electron acceptor, electron transfer flavoproteinubiquinone oxidoreductase (ETF-QO), cause the human inherited metabolic disease glutaric acidemia type II. In this disease, electron transfer from nine primary flavoprotein dehydrogenases to the main respiratory chain is impaired. Among these dehydrogenases are the four chain length-specific flavoprotein dehydrogenases of fatty acid ␤-oxidation. In this investigation, two mutations in the ␣ subunit that have been identified in patients were expressed in Escherichia coli. Of the two mutant alleles, ␣T266M and ␣G116R, the former is the most frequent mutation found in patients with ETF deficiency. The crystal structure of human ETF shows that ␣G116 lies in a hydrophobic pocket, under a contact residue of the ␣/␤ subunit interface, and that the hydroxyl hydrogen of ␣T266 is hydrogen Stable expression of the ␣G116R ETF required coexpression of the chaperonins, GroEL and GroES. ␣G116R ETF folds into a conformation different from the wild type, and is catalytically inactive in crude extracts. It is unstable and could not be extensively purified. The ␣T266M ETF was purified and characterized after stabilization to proteolysis in crude extracts. Although the global structure of this mutant protein is unchanged, its flavin environment is altered as indicated by absorption and circular dichroism spectroscopy and the kinetics of flavin release from the oxidized and reduced protein.The loss of the hydrogen bond at N(5) of the flavin and the altered flavin binding increase the thermodynamic stability of the flavin semiquinone by 10-fold relative to the semiquinone of wild type ETF. The mutation has relatively little effect on the reductive half-reaction of ETF catalyzed by sarcosine and medium chain acyl-CoA dehydrogenases which reduce the flavin to the semiquinone. However, k cat /K m of ETF-QO in a coupled acylCoA:ubiquinone reductase assay with oxidized ␣T266M ETF as substrate is reduced 33-fold; this decrease is due in largest part to a decrease in the rate of disproportionation of the ␣T266M ETF semiquinone catalyzed by ETF-QO. Electron transfer flavoproteins (ETF)1 are heterodimeric, FAD-containing proteins that transfer electrons between primary dehydrogenases and respiratory chains in eukaryotic and prokaryotic cells. In mammalian systems, ETF transfers electrons from nine mitochondrial flavoprotein dehydrogenases to the main respiratory chain via the iron-sulfur flavoprotein, ETF-ubiquinone oxidoreductase (ETF-QO) (1). Porcine ETF is apparently closely related to human ETF (2, 3). Both proteins stabilize an anionic flavin semiquinone upon reduction by the flavoprotein dehydrogenases (4, 5). Reduction of ETF to the hydroquinone oxidation state by the dehydrogenases is very slow and not kinetically significant (4, 6). However, kinetic studies of the ETF-QO-catalyzed reduction of ubiquinone by reduced ETF by Ramsay et al. indicated that ETF hydroquinone, and not the semiquinone, is the reductant of ubiquinone (6). Thi...

The diagnosis of inborn errors of metabolism (IEM) takes many forms. Due to the implementation and advances in newborn screening (NBS), the diagnosis of many IEM has become relatively easy utilizing laboratory biomarkers. For the majority of IEM, early diagnosis prevents the onset of severe clinical symptoms, thus reducing morbidity and mortality. However, due to molecular, biochemical, and clinical variability of IEM, not all disorders included in NBS programs will be detected and diagnosed by screening alone. This article provides a general overview and simplified guidelines for the diagnosis of IEM in patients with and without an acute metabolic decompensation, with early or late onset of clinical symptoms. The proper use of routine laboratory results in the initial patient assessment is also discussed, which can help guide efficient ordering of specialized laboratory tests to confirm a potential diagnosis and initiate treatment as soon as possible.

We report two women with glutaric acidemia type I in whom the diagnosis was unsuspected until a low carnitine level was found in their newborn children. Both mothers had low carnitine in plasma. In the first, organic acid analysis was only done after fibroblast studies revealed normal carnitine uptake. Having learned from the first family, organic acid analysis was done immediately in the mother of family 2. In both, the plasma acylcarnitine profile was normal but both excreted the metabolites typical of their disorder. One of the women was a compound heterozygote for distinct mutations in the glutaric acid dehydrogenase gene, whereas the second was either homozygous or hemizygous for a mutation in Exon 6 of the gene.