ETF dehydrogenase

The oxidation of fatty acids is catalyzed by the FAD-containing acyl coenzyme A dehydrogenases which transfer reducing equivalents to the mitochondrial respiratory chain via a flavin-containing electron transfer flavoprotein (ETF) and subsequently via an ETF dehydrogenase (an Fe/S flavoprotein In addition to the mammalian... 

The mitochondrial respiratory chain, which contains at least 13 Fe-S clusters (Figure 6), perhaps best illustrates the importance of Fe-S clusters in membrane-bound electron transport. Electrons enter via three principal pathways, from the oxidation of NADH to NAD+ (NADH-ubiquinone oxidoreductase or Complex I) and succinate to fumarate (succinate ubiquinone oxidoreductase or Complex II), and from the /3-oxidation of fatty acids via the electron transferring flavoprotein (ETF-ubiquinone oxidoreductase). All three pathways involve a complex Fe S flavoprotein dehydrogenase, that is, NADH dehydrogenase, succinate dehydrogenase, and ETF dehydrogenase, and in each case the Fe-S clusters mediate electron transfer from the flavin active site to the ubiquinone pool via protein-associated ubiquinone. 

Defects of fatty acid catabolism, with the exception of SCAD deficiency, generally have elevation of more than one characteristic metabolite. MCAD deficiency is characterized by accumulation of C6, C8 (mainly) and C10 l species. LCAD and VLCAD are characterized by accumulation of C14 l, C14 2 and (usually) C16 and C18 l species. LCHADD and TFP deficiencies are characterized by the accumulation of OH-C16, 0H-C18 1 and usually at least one of the other long-chain species C14 1, C16 and C18 l. The CPT-II and CAT (carnitine/acylcarnitine translocase) deficiencies are characterized by marked elevation of both C16 and C18 1, but not C14 1. Multiple acyl-CoA deficiency (MAD) has several different etiologies, including electron transferring protein (ETF) deficiency, ETF-dehydrogenase deficiency and riboflavin deficiency. Disease patterns vary considerably. In severe forms of the disorder, a generalized marked elevation of mxiltiple intermediates is observed. CPT-I should be suspected when both C16 and Cl8 1 are very low in whole blood, especially if free carnitine is normal or elevated. 

Apart from glutaric aciduria II (ETF or ETF dehydrogenase deficiency, see Sect. 14.10) a third disorder with peroxisomal glutaryl-CoA oxidase deficiency leading to glutaric aciduria has been described (glutaric aciduria III, see Chap. 24). 

I These dehydrogenases are lim-j ited to the respiratory chain at 7 the level of complex III by ETF (5) dehydrogenase (6) and ubiqui-... 

Table 3.2.5 Disorders detectable by the in vitro probe assay. ETF Electron transfer flavoprotein, MADD multiple acyl-CoA dehydrogenase deficiency...

Each of the forms of ETF isolated from the different sources contain FAD as coenzyme and form an anionic semiquinone on one-electron reduction. Stopped-flow kinetic studies on the pig liver ETF showed the anionic flavin semiquinone to be formed at times faster than catalytic turnover and thus demonstrate the participation of the anionic FAD semiquinone as an intermediate in the acceptance of reducing equivalents from the dehydrogenase. These studies would also imply the intermediacy of the semiquinone form of the acyl CoA dehydrogenase which would have been expected to form a neutral flavin semiquinone at the time the studies of Hall and Lambeth were performed, however, no spectral evidence for its formation were found. Recent studies have shown that the binding of CoA analogs to the dehydrogenase results in the perturbation of the pKa of the FAD semiquinone such that an anionic (red) rather than the neutral (blue) semiquinone is formed. This perturbation was estimated to reduce the pKa by at least 2.5 units to a value of... 

These data would suggest that both the dehydrogenase and ETF form anionic flavin semiquinone on electron transfer which cannot be differentiated spectrally, and thus the kinetic intermediacy of the anionic flavin semiquinone in electron transfer between the dehydrogenase and ETF may be due to both flavoproteins. 

The properties of the semiquinone from of the ETF isolated from the methylotrophic bacterium resemble those of the bacterial flavodoxins with the exception that flavodoxins form neutral semiquinones whereas this ETF forms an anionic semiquinone. Nearly quantitative anionic semiquinone formation is observed either in the presence of excess dithionite or when excess trimethylamine and a catalytic amount of trimethylamine dehydrogenase are added. Of interest is the apparent stability of the anionic semiquinone towards oxidation by O2 but not to oxidizing agents such as ferricyanide. This appears to be the first example of an air-stable protein-bound anionic flavin semiquinone. Future studies on the factors involved in imparting this resistance to O2 oxidation by the apoprotein are looked forward to with great interest. 

Each molecule of FADH2 formed during oxidation of the fatty acid donates a pair of electrons to ETF of the respiratory chain, and about 1.5 molecules of ATP are generated during the ensuing transfer of each electron pair to O2. Similarly, each molecule of NADH formed delivers a pair of electrons to the mitochondrial NADH dehydrogenase, and the subsequent transfer of each pair of electrons to O2 results in formation of about 2.5 molecules of ATP. Thus four molecules of ATP are formed for each two-carbon unit removed in one pass through the sequence. Note that water is also produced in this... 

Fig. 5.2. Possible metabolic pathways in facultative anaerobic mitochondria. Shaded boxes show components of the electron-transport chain used during hypoxia, open boxes are components used during aerobiosis, and the hatched boxes (complex I and ATP-synthase) are components used under aerobic as well as anaerobic conditions. ASCT acetate succinate CoA-transferase, C cytochrome c, Cl, CIII and CIV complexes I, III and IV of the respiratory chain, CITR citrate, ECR enoyl-CoA reductase (such as present in Ascaris suum), ETF electron-transfer flavoprotein, ETF RQ OR electron-transfer flavoproteimrhodoquinone oxidoreductase, FRD fumarate reductase, FUM fumarate, MAE malate, OXAC oxaloacetate, PYR pyruvate, RQ rhodoquinone, SDH succinate dehydrogenase, SUCC succinate, Succ-CoA succinyl-CoA, TER trans-2-enoyl-CoA reductase (such as present in E. gracilis), UQ ubiquinone...

Figure 18.5 The glycerol-3-phosphate shuttle. This shuttle is used to bring electrons from cytosolic NADH into mitochondria. The mitochondrial glycerol-3-phosphate dehydrogenase with its FAD prosthetic group is bound to the inner mitochondrial membrane. ETF is electron transfer flavoprotein, which extracts electrons from the FADH2 of mitochondrial glycerol-3-phosphate dehydrogenase and with it reduces ubiquinone (UQ).

We now begin upon an oxidation phase With FAD-dependent dehydrogenase (The flavoprotein is oxidised again By ETF, thence by the chain). 

A distinct electron transfer flavoprotein (ETF) is the single-electron acceptor for a variety of flavoprotein dehydrogenases, including acyl CoA, glutaryl CoA, sarcosine, and dimethylglycine dehydrogenases. It then transfers the electrons to ETF-ubiquinone reductase, the iron-sulfur flavoprotein that reduces ubiquinone in the mitochondrial electron transport chain. 

The foeus of this chapter is the soluble electron transfer complex formed between the nieotinamide-independent trimethylamine dehydrogenase (TMADH) and eleetron transferring flavoprotein (ETF). Recent studies of this physiological electron transfer complex have provided invaluable insight into (i) the mechanisms of inter and intraprotein electron transfer between flavin and Fe/S centers, (ii) the role of dynamics in interprotein electron transfer and (hi) quantum meehanieal mechanisms for the cleavage of substrate C-H bonds and the subsequent transfer of reducing equivalents to flavin redox centers. Brief mention is made of early structural and cofactor analyses for this redox system, but more detailed accounts of this work can be found in earlier reviews on the subjeet (e.g. Steenkamp and Mathews, 1992). 

Fig. 3.1. A, The respiratory chain. Q and c stand for ubiquinone and cytochrome c, respectively. Auxiliary enzymes that reduce ubiquinone include succinate dehydrogenase (Complex II), a-glycerophosphate dehydrogenase and the electron-transferring flavoprotein (ETF) of fatty acid oxidation. Auxiliary enzymes that reduce cytochrome c include sulphite oxidase. B, Thermodynamic view of the respiratory chain in the resting state (State 4). Approximate values are calculated according to the Nernst equation using oxidoreduction states from work by Muraoka and Slater, (NAD, Q, cytochromes c c, and a oxidation of succinate [6]), and Wilson and Erecinska (b-562 and b-566 [7]). The NAD, Q, cytochrome b-562 and oxygen/water couples are assumed to equilibrate protonically with the M phase at pH 8 [7,8]. E j (A ,/ApH) for NAD, Q, 6-562, and oxygen/water are taken as —320 mV ( — 30 mV/pH), 66 mV (- 60 mV/pH), 40 mV (- 60 mV/pH), and 800 mV (- 60 mV/pH) [7-10]. FMN and the FeS centres of Complex I (except N-2) are assumed to be in redox equilibrium with the NAD/NADH couple, FeS(N-2) with ubiquinone [11], and cytochrome c, and the Rieske FeS centre with cytochrome c [10]. The position of cytochrome a in the figure stems from its redox state [6] and its apparent effective E -, 285 mV in...

Glutaric acidemia type II is caused by defects in the ETF/ETF-QO proteins. The clinical manifestations of these disorders are similar to medium-chain acyl-CoA dehydrogenase deficiency (discussed later). The double bond formed by the acyl-CoA dehydrogenase has a trans configuration. The double bonds in naturally occurring fatty acids are generally in the cis configuration. The oxidation of unsaturated m-fatty acids requires two auxiliary enzymes, enoyl-CoA isomerase and 2,4-dienoyl-CoA reductase. 

Figure 8. ESR (upper) and ENDOR (lower) spectra from flavoproteins at — 160°C. a, Semiquinone radicals from the one-electron reduction of electron-transfer flavoprotein (ETF) b, semiquinone radicals from the one-electron reduction of sarcosine dehydrogenase. From [144], with permission.

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