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PubMed1982

FMN phosphatase and FAD pyrophosphatase in rat intestinal brush borders: role in intestinal absorption of dietary riboflavin.

Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are two major coenzyme forms of dietary riboflavin. Yet little attention has been given to the release of the vitamin from its coenzyme forms during the absorptive process. Homogenates from rat intestine catalyze the hydrolases of these flavin coenzymes. To determine the location of FMN and FAD hydrolases, homogenates of intestinal mucosa were fractioned. FMN and FAD phosphatases were localized in brush border membranes. FAD pyrophosphatase activity was maximal at pH between 6.5 and 8.5 while FMN phosphatase has a pH optimum of 7.5 - 8.0. FAD pyrophosphatase is more stable to heat. The two enzymes separate on ion exchange chromatography of an isobutanol extract of intestinal brush border membrane fraction. Inhibition of 14C-riboflavin uptake by FMN and FAd in everted rings of rat intestine is directly related to the amount of conversion of these coenzymes to free riboflavin by intestinal enzymes. When FMN and FAD conversion to riboflavin is inhibited by EDTA, competition with 14C-riboflavin for transport was correspondingly decreased. These studies are best explained by a sequential process in which hydrolysis of FMN and FAD by enzymes of the intestinal brush border is followed by absorption of free riboflavin.

Animals↗
PubMed2006

Conversion of FAD to FMN and riboflavin in plasma: effects of measuring method.

The stability of flavin adenin dinucleotide (FAD) in plasma was studied under a low-intensity light and FAD was found to be converted to flavin mononucleotide (FMN) and riboflavin (RF) in both human and rat plasma. The hydrolysis rates of FAD in plasma at 4 degrees C were lower than those at 37 degrees C. In addition, the hydrolysis rates were markedly inhibited when EDTA, known as an anticoagulant, was added to plasma. These results indicated that plasma samples in pharmacokinetic studies should be pretreated with EDTA, extracted at the earliest convenience and lower temperature like 4 degrees C to keep a high stability. The pharmacokinetic study after intravenous administration of FAD at a dose of 500 nmol/kg as FAD in rats was performed with plasma samples after addition of EDTA under strict light and temperature control. A measurable amount of FAD in plasma together with rapid conversions of FAD to FMN and RF were observed in rat plasma. The AUC values (mean+/-S.D. of 4 rats) for FAD, FMN and RF were 707+/-378, 3643+/-958 and 30095+/-3544 nmol x min/l, respectively. Using excess EDTA under strict temperature and light control may be useful for assessment of vitamin B2 in the in vivo study.

Animals↗
PubMed1995

Nucleotide sequence of the FAD synthetase gene from Corynebacterium ammoniagenes and its expression in Escherichia coli.

The nucleotides of a bifunctional enzyme FAD synthetase gene, which showed both flavokinase and ATP:FMN adenylyltransferase activities, from Corynebacterium ammoniagenes were sequenced. The FAD synthetase gene product consisted of 338 amino acids and had a calculated molecular weight of 37,712. The deduced protein sequence of the FAD synthetase shared a homology with those of the protein X of Escherichia coli, which has been reported to have both flavokinase and ATP:FMN adenylyltransferase activities like the FAD synthetase of C. ammoniagenes, and the protein X of Pseudomonas fluorescens. From the analysis of the flanking sequences of the FAD synthetase gene, the gene organization and the operon structure around the FAD synthetase gene of C. ammoniagenes were thought to be different from those of Gram-negative bacteria. An over-expression system of the FAD synthetase of C. ammoniagenes was constructed in E. coli to study the structure and function of the protein. Under the tandem tryptophan promoter, the FAD synthetase activity increased 2231 times compared to that of non-transformed C. ammoniagenes.

Amino Acid Sequence↗
PubMed2000

Activation domain in P67phox regulates the steady state reduction of FAD in gp91phox.

An activation domain in p67(phox) (residues 199-210) is critical for regulating NADPH oxidase activity in cell-free system [10] To determine the steady state reduction of FAD, thioacetamide-FAD was reconstituted in gp91(phox), and the fluorescence of its oxidised form was monitored. Omission of p67(phox) decreased the steady state reduction of the FAD from 28% to 4%, but omission of p47(phox) had little effect. A series of the truncated forms of p67(phox) were expressed in E.coli to determine the domain in p67(phox) which is essential for regulating the steady state of FAD reduction. The minimal length of p67(phox) for for regulating the steady state of FAD reduction is shown to be 1-210 using a series of truncation mutants which indicates that the region 199-210 is also important for regulating electron flow within flavocytochrome b(558). The deletion of this domain not only decreased the superoxide generation but also decreased the steady state of FAD reduction. Therefore, the activation domain on p67(phox) regulates the reductive half-reaction for FAD, consistent with a dominant effect on hydride/electron transfer from NADPH to FAD.

Amino Acid Sequence↗
PubMed1988

5'-Nucleotidase of human placental trophoblastic microvilli possesses cobalt-stimulated FAD pyrophosphatase activity.

An enzyme with FAD pyrophosphatase activity was extracted from human placental syncytiotrophoblast microvilli and purified to near-homogeneity. The enzyme has been identified as 5'-nucleotidase by several criteria. Throughout purification, parallel increases in the specific activities of FAD pyrophosphatase and AMP phosphatase were observed. The enzyme was a glycoprotein with a subunit molecular weight of 74,000. EDTA treatment resulted in a marked decline in both activities, and restoration of FAD pyrophosphatase activity but not 5'-nucleotidase activity was accomplished by the addition of Co2+ or, to a lesser extent, Mn2+. The substrate specificity of the 5'-nucleotidase activity that we observed agreed closely with the results of others. The pyrophosphatase activity was relatively specific for FAD. ADP, ATP, NAD(H), and FMN were not hydrolyzed, and ADP strongly inhibited both activities. For FAD pyrophosphatase activity, a Km of 1.2 x 10(-5) M and a Vmax of 1.1 mumol/min/mg protein were determined in assays performed in the presence of Co2+. In the absence of added Co2+, the Vmax declined but the Km was unchanged. For 5'-nucleotidase (AMP as substrate) the Km was 4.1 x 10(-5) M and the Vmax 109 mumol/min/mg protein. Hydrolysis of FMN to riboflavin was observed in partially purified detergent extracts of microvilli that contained alkaline phosphatase activity and lacked FAD pyrophosphatase and 5'-nucleotidase activity. The presence of both FAD pyrophosphatase and FMN phosphatase activities in syncytiotrophoblast microvilli supports the view that the placental uptake of vitamin B2 involves the hydrolysis of FAD and FMN to riboflavin which is then absorbed, a sequence postulated for intestinal absorption and liver uptake.

5'-Nucleotidase↗
PubMed1983

Mechanistic studies of p-hydroxybenzoate hydroxylase reconstituted with 2-Thio-FAD.

2-Thio-FAD (oxygen substituent at position 2 is replaced by sulfur) was used to reconstitute the apoenzyme of p-hydroxybenzoate hydroxylase. The 2-thio-FAD enzyme differs from native enzyme in several respects. While the native enzyme catalyzes the fully coupled hydroxylation of p-hydroxybenzoate, the 2-thio-FAD enzyme shows no hydroxylation of this substrate, instead reducing molecular oxygen to hydrogen peroxide. The rate of reduction of 2-thio-FAD p-hydroxybenzoate hydroxylase by NADPH in the presence of substrate was 7-fold faster than with the native enzyme. However, the oxygen reactivity of the reduced 2-thio-FAD enzyme was less than 1% that of native enzyme. This slow oxygen reaction results in the very high KmO2 observed in steady state kinetic studies of the modified enzyme. Stopped flow studies of the oxygen reaction of the reduced 2-thio-FAD enzyme in the presence of substrate confirmed the formation of a transient intermediate. The spectrum of this intermediate is very similar to those of the flavin-C(4a) adducts obtained with 2-thio-FMN lactate oxidase. This evidence suggests that reduced 2-thio-FAD p-hydroxybenzoate hydroxylase forms a flavin-C(4a)-hydroperoxide on reaction with oxygen in a reaction analogous to that with native enzyme, but that the resulting peroxyflavin is incompetent as an oxygenating species, breaking down instead to oxidized 2-thio-FAD enzyme and hydrogen peroxide.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1996

Characterization of the FAD binding domain of cytochrome P450 reductase.

The redox potentials of FAD and FMN of Cytochrome P450 reductase (reductase) are equivalent in solution but differ by 138 mV when bound to reductase. The interaction of each flavin with its flavin binding domain confers the unique electron transferring abilities to each flavin. In order to determine flavin binding properties and activity of the FAD binding domain, we have expressed in pTrcHis three fragments (1161, 1244, and 1556 bp) of rat liver reductase cDNA encompassing the proposed FAD and NADPH binding domain. The FAD binding fragments from cells harboring the 1161- and 1556-bp-containing vectors were stable and bound 0.66 and 0.71 mol FAD/mol enzyme, respectively. Both fragments reduce ferricyanide (54 and 104% of FMN-less reductase/mol bound flavin, respectively) and participate in the transhydrogenation reaction of 3-AcPy-ADP (41 and 65% of FMN-less reductase/mol bound flavin, respectively). FAD-less fragments were purified and reconstituted with 8-amino-FAD and 8-chloro-FAD to determine binding efficiencies.

Animals↗
PubMed1989

Site-directed mutagenesis of the FAD-binding histidine of 6-hydroxy-D-nicotine oxidase. Consequences on flavinylation and enzyme activity.

In 6-hydroxy-D-nicotine oxidase (6-HDNO) FAD is covalently bound to His71 of the polypeptide chain by an 8 alpha-(N3-histidyl)-riboflavin linkage. The FAD-binding histidine was exchanged by site-directed mutagenesis to either a Cys- or Tyr-residue, two amino acids known to be involved in covalent binding of FAD in other enzymes, or to a Ser-residue. None of the amino acid replacements for His71 allowed covalent FAD incorporation into the 6-HDNO polypeptide. Thus, the amino acid residues involved in covalent FAD-binding require a specific polypeptide surrounding in order for this modification to proceed and cannot be replaced with each other. Enzyme activity was completely abolished with Tyr in place of His71. 6-HDNO activity with non-covalently bound FAD was found with 6-HDNO-Cys and to a lesser extent also with 6-HDNO-Ser. However, the Km values for 6-HDNO-Cys and 6-HDNO-Ser were increased approximately 20-fold as compared to 6-HDNO-His. Both mutant enzymes, in contrast to the wild-type enzyme, needed additional FAD in the enzymatic assay (50 microM for 6-HDNO-Ser and 10 microM for 6-HDNO-Cys) for maximal enzyme activity.

Base Sequence↗
PubMed2006

Over-expression in Escherichia coli and characterization of two recombinant isoforms of human FAD synthetase.

FAD synthetase (FADS) (EC 2.7.7.2) is a key enzyme in the metabolic pathway that converts riboflavin into the redox cofactor FAD. Two hypothetical human FADSs, which are the products of FLAD1 gene, were over-expressed in Escherichia coli and identified by ESI-MS/MS. Isoform 1 was over-expressed as a T7-tagged protein which had a molecular mass of 63kDa on SDS-PAGE. Isoform 2 was over-expressed as a 6-His-tagged fusion protein, carrying an extra 84 amino acids at the N-terminal with an apparent molecular mass of 60kDa on SDS-PAGE. It was purified near to homogeneity from the soluble cell fraction by one-step affinity chromatography. Both isoforms possessed FADS activity and had a strict requirement for MgCl(2), as demonstrated using both spectrophotometric and chromatographic methods. The purified recombinant isoform 2 showed a specific activity of 6.8+/-1.3nmol of FAD synthesized/min/mg protein and exhibited a K(M) value for FMN of 1.5+/-0.3microM. This is the first report on characterization of human FADS, and the first cloning and over-expression of FADS from an organism higher than yeast.

Amino Acid Sequence↗
PubMed2006

Dodecin sequesters FAD in closed conformation from the aqueous solution.

Both extensive theoretical calculations and experimental data obtained during several decades leave little doubt that flavin adenine dinucleotide (FAD) exists in an open as well as in a closed conformation in aqueous solution. However, the knowledge about the intramolecularly stacked complex of FAD is constructed on indirect methods while direct structural evidence is lacking. Recently, dodecin was reported as an unspecific flavin binding protein which exhibits the unique binding mode of incorporating stacked dimers of flavins into a single binding pocket. Here, we show that FAD is not bound in this manner, but in monomers of intramolecularly stacked conformation. As resulting from the dodecin ligand binding characteristic, this FAD stacked conformation suggests to be directly sequestered from the aqueous solution and thus to be the first X-ray structural view on a FAD solution-stacked form. Moreover, in extraordinary FAD binding, dodecin serves as a model for studying bound monomeric (FAD) versus bound dimeric (e.g. riboflavin) flavin properties.

Binding Sites↗
PubMed1984

FAD requirement for the reduction of coenzyme F420 by hydrogenase from Methanobacterium formicicum.

Hydrophobic interaction chromatography of coenzyme F420-reducing hydrogenase purified from Methanobacterium formicicum depleted protein-bound FAD and eliminated the ability to reduce coenzyme F420. Preincubation of the FAD-depleted hydrogenase with FAD restored 85% of the coenzyme F420-reducing activity. FMN did not replace FAD. A Kd of 12 microM was estimated for FAD. Analysis of the reactivated hydrogenase following molecular sieve column chromatography showed that FAD was bound to protein. The results indicate that protein-bound FAD is reversibly removed from the coenzyme F420-reducing hydrogenase and that this flavin is required for the reduction of coenzyme F420.

Chromatography↗
PubMed1995

Mutagenesis at a highly conserved tyrosine in monoamine oxidase B affects FAD incorporation and catalytic activity.

Monoamine oxidase B (MAO B), an integral protein of the outer mitochondrial membrane, catalyzes the oxidative deamination of various neuroactive and vasoactive amines. A covalently bound FAD cofactor at Cys-397 of human MAO B is required for the oxidation of the amine substrates. In addition to the covalent binding site, MAO B also contains a noncovalent FAD binding region (residues 6-34) known as the dinucleotide binding motif. Previously, we have shown that Glu-34 is required for catalytic activity, presumably by forming a hydrogen bond between the carboxylate group of glutamate and the 2'-hydroxyl group of ribose in the AMP moiety of FAD. In this work, we have identified a third FAD binding site in MAO B (residues 39-46) by sequence comparisons to other flavoenzymes. The conserved sequence contains a tyrosine residue (Tyr-44) which, based on the X-ray crystal structure of ferredoxin-NADP+ reductase, is postulated to participate in FAD binding through van der Waals contact with the isoalloxazine ring and a hydrogen bond to the 3'-hydroxy of the ribityl moiety. To test the postulated role of this tyrosine residue, site-directed mutants that encode substitutions at Tyr-44 were prepared and expressed in mammalian COS-7 cells. Variant MAO B enzymes were then characterized with respect to enzymatic activity and [14C]FAD incorporation. Substitution of tyrosine with phenylalanine had no effect on MAO B activity or the level of [14C]FAD incorporation compared to the wild-type enzyme, indicating that the hydroxyl group of the tyrosine residue was not essential at residue 44.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2005

Light-induced structural changes of apoprotein and chromophore in the sensor of blue light using FAD (BLUF) domain of AppA for a signaling state.

AppA is a new class blue-light receptor controlling photosynthesis gene expression in the purple bacterium Rhodobacter sphaeroides and retains a characteristic flavin adenine dinucleotide (FAD)-binding domain named the "sensor of blue light using FAD" (BLUF). AppA functions as an antirepressor controlling transcription of photosynthesis genes through the direct association with a transcriptional repressor PpsR in a blue-light-dependent manner [Masuda and Bauer (2002) Cell 110, 613-623]. Illumination of AppA induces a red shift in the UV-visible absorption of FAD, which results in a signaling state of AppA. Light-induced Fourier transform infrared (FTIR) difference spectrum of the AppA BLUF domain showed relatively simple features, which were mainly composed of two sets of derivative-shaped sharp bands at 1709(-)/1695(+) and 1632(+)/1619(-) cm(-)(1). We have developed an in vitro reconstitution method, by which a fully functional BLUF domain was reconstituted from free FAD and an apoprotein for the BLUF domain of AppA. An AppA BLUF domain that consisted of an apoprotein isotopically labeled with (13)C and unlabeled FAD was constituted using this method, and hydrated and deuterated samples were applied to FTIR spectroscopic analyses. When the spectra for the reconstituted domain were compared with those for uniformly (15)N- and (13)C-labeled or deuterated domains as well as for the unlabeled domain, the IR bands responsible for the light-induced changes in the FAD chromophore and apoprotein were identified. Unexpectedly, the light-induced spectrum of the unlabeled BLUF domain of AppA was predominantly composed of multiple apoprotein bands, while a C(4)=O stretching of an isoalloxazine ring was the only band exclusively assigned to FAD. The results showed that relatively large structural changes occur in the protein backbone of the BLUF domain of AppA upon illumination. These changes were discussed in relation to the mechanistic role of the BLUF domain in the process of blue-light perception by AppA.

Apoproteins↗
PubMed2004

Mutagenesis study of the 2Fe-2S center and the FAD binding site of the Na(+)-translocating NADH:ubiquinone oxidoreductase from Vibrio cholerae.

Many marine and pathogenic bacteria have a unique sodium-translocating NADH:ubiquinone oxidoreductase (Na(+)-NQR), which generates an electrochemical Na(+) gradient during aerobic respiration. Na(+)-NQR consists of six subunits (NqrA-F) and contains five known redox cofactors: two covalently bound FMNs, one noncovalently bound FAD, one riboflavin, and one 2Fe-2S center. A stable neutral flavin-semiquinone radical is observed in the air-oxidized enzyme, while the NADH- or dithionite-reduced enzyme exhibits a stable anionic flavin-semiquinone radical. The NqrF subunit has been implicated in binding of both the 2Fe-2S cluster and the FAD. Four conserved cysteines (C70, C76, C79, and C111) in NqrF match the canonical 2Fe-2S motif, and three conserved residues (R210, Y212, S246) have been predicted to be part of a flavin binding domain. In this work, these two motifs have been altered by site-directed mutagenesis of individual residues and are confirmed to be essential for binding, respectively, the 2Fe-2S cluster and FAD. EPR spectra of the FAD-deficient mutants in the oxidized and reduced forms exhibit neutral and anionic flavo-semiquinone radical signals, respectively, demonstrating that the FAD in NqrF is not the source of either radical signal. In both the FAD and 2Fe-2S center mutants the line widths of the neutral and anionic flavo-semiquinone EPR signals are unchanged from the wild-type enzyme, indicating that neither of these centers is nearby or coupled to the radicals. Measurements of steady-state turnover using NADH, Q-1, and the artificial electron acceptor ferricyanide strongly support an electron transport pathway model in which the noncovalently bound FAD in the NqrF subunit is the initial electron acceptor and electrons then flow to the 2Fe-2S center.

Amino Acid Sequence↗
PubMed1982

The association of FAD with the cytochrome b-245 of human neutrophils.

A plasma membrane fraction prepared from human neutrophils had a fluorescence resembling that of a fluorescent flavoprotein, with emission maximum near 520nm and excitation maxima near 380 and 460nm. The fluorescence emission and excitation properties of Triton N-101-solubilized membrane fraction resembled those of FAD. FAD was present in the membranes at a concentration of 417pmol/mg of protein and cytochrome b(-245) at a concentration of 407pmol/mg of protein. In a 110-fold purified preparation of cytochrome b(-245) the ratio of FAD:cytochrome b was 1:1. Analytical gradient centrifugation of neutrophil homogenates shows a coincidence of two cytochrome b peaks and two peaks of fluorescence, corresponding with plasma membrane and specific granule fractions; most of the FAD was non-fluorescent and located in fractions lighter than the plasma membrane. Plasma membrane fractions prepared from neutrophils of patients suffering from the X-linked form of chronic granulomatous disease lacked cytochrome b and contained 194pmol of FAD/mg of protein; plasma membrane fractions prepared from neutrophils of patients with the autosomal recessive form of chronic granulomatous disease contained both cytochrome b(-245) and FAD in the normal range of concentrations in a ratio of 1:1. Phagocytic vesicles were prepared from normal neutrophils and found to contain FAD and cytochrome b in a ratio 2.22:1, suggesting that activation of neutrophils many involve the incorporation of an additional flavin into the membrane. Under anaerobic conditions in the presence of EDTA to act as an electron donor to a flavin, the cytochrome b(-245) of neutrophil membranes was partly (12%) photoreducible, an effect increased to 100% by the addition of FMN. The extent of reduction of cytochrome b in an anaerobic neutrophil homogenate containing NADH increased from 30% to 70% on illumination. We suggest that these results indicate a close association between FAD and cytochrome b(-245) and support a scheme for electron transport thus: [Formula: see text]

Cell Membrane↗
PubMed1992

Cytochrome b-245 is a flavocytochrome containing FAD and the NADPH-binding site of the microbicidal oxidase of phagocytes.

The NADPH oxidase of phagocytic cells is important for the efficient killing and digestion of ingested microbes. A very unusual low-potential cytochrome b (b-245) is the only redox molecule to have been identified in this system. The FAD-containing flavoprotein that binds NADPH and transfers electrons to the cytochrome has eluded identification for three decades. We show here that the haem/FAD ratio in the membranes does not change significantly on activation of this oxidase, indicating that the FAD is present in the membranes from the outset and not recruited from the cytosol. The FAD content of membranes from cells of patients with X-linked chronic granulomatous disease (CGD) lacking the cytochrome b was roughly one-quarter of that in normal subjects and in autosomal recessive CGD patients lacking the cytosolic protein p47-phox. Similar low amounts of FAD were present in uninduced promyelocytic (HL60) cells, suggesting that the low amount of FAD in cells from X-CGD patients was probably unrelated to this oxidase system. Cytochrome b-245 appears to bind both the haem and FAD, in a molar ratio of 2:1. The e.p.r. signal of the purified cytochrome was weak and had an asymmetric g(z) peak at g = 3.31. The purified cytochrome could be partially reflavinated (about 20%) in the presence of lipid. Amino acid sequence homology was detected between the beta-subunit of this cytochrome b and the ferredoxin-NADP+ reductase (FNR) family of reductases in the putative NADPH- and FAD-binding sites. 32P-labelled 2-azido-NADP was used as a photoaffinity label for the NADPH-binding site. Labelling that was competed off with NADP was observed in the region of the beta-subunit of the cytochrome. No labelling was seen in this region in X-CGD in three subjects in whom this cytochrome was missing and in a third in whom it was present but bore a Pro-His transposition in the putative NADPH-binding site. These studies indicate that cytochrome b-245 is a flavocytochrome, the first described in higher eukaryotic cells, bearing the complete electron-transporting apparatus of the NADPH oxidase.

Amino Acid Sequence↗
PubMed2005

Single amino acid exchanges in FAD-binding domains of squalene epoxidase of Saccharomyces cerevisiae lead to either loss of functionality or terbinafine sensitivity.

Squalene epoxidase (Erg1p) is an essential enzyme in the ergosterol biosynthesis pathway in yeast. For its enzymatic activity, Erg1p requires molecular oxygen, NAD(P)H and FAD. Amino acid analysis and sequence alignment with other squalene epoxidases revealed two highly conserved FAD-binding domains, FAD I and FAD II. By random PCR mutagenesis of the ERG1 gene, one erg1 allele was isolated that carries a mutation leading to a single amino acid exchange in the FAD I domain close to the N-terminus of Erg1p. This erg1 allele codes for functional squalene epoxidase and renders yeast cells hypersensitive to terbinafine. Amino acid exchanges of other conserved residues in the FAD I and FAD II regions either led to non-functional squalene epoxidase or to the formation of squalene epoxidase with wild-type properties. These results describe the importance of specific amino acids for enzymatic activity in the yeast squalene epoxidase Erg1p.

Amino Acid Sequence↗
PubMed1995

Isoalloxazine ring of FAD is required for the formation of the core in the Hsp60-assisted folding of medium chain acyl-CoA dehydrogenase subunit into the assembly competent conformation in mitochondria.

We studied the role of FAD in the intramitochondrial folding and assembly of medium-chain acyl-CoA dehydrogenase (MCAD), a homotetrameric mitochondrial enzyme containing a molecule of non-covalently bound FAD/monomer. In the MCAD molecule, FAD is buried in a crevice containing the active center. We have previously shown that upon import into mitochondria, newly processed MCAD is first incorporated into a high molecular weight (hMr) complex and that the hMr complex mainly consisted of MCAD-heat-shock protein 60 (hsp60) complex (Saijo, T., Welch, W.J., and Tanaka, K (1994) J. Biol. Chem. 269, 4401-4408). In the present study, we incubated in vitro synthesized precursor MCAD with mitochondria isolated from normal and riboflavin-deficient rat liver for 10-60 min and fractionated the solubilized mitochondria using gel filtration. The amount of MCAD in the hMr complex was larger and that of tetramer was smaller in riboflavin-deficient mitochondria than in control at any time point. In addition, riboflavin-deficient mitochondria were solubilized after 10-min import in a buffer containing ATP and were chased in the presence of FAD, FMN, or NAD+ or without any addition. The mitochondrial proteins were analyzed using gel filtration or immunoprecipitated with anti-hsp60 antibody. After 60-min chase in the presence of FAD, the majority of MCAD in the complex with hsp60 was transferred to tetramer, whereas no such transfer occurred after the chase in the absence of FAD. When chase was done in the presence of FMN, a significant amount of MCAD was transferred from the complex with hsp60 to tetramer, but the transfer was not as efficient as in the presence of FAD. The chase in the presence of NAD+ resulted in no transfer. These data suggest that isoalloxazine ring of FAD plays a critical role, exerting nucleating effect, in the hsp60-assisted folding of MCAD subunit into an assembly competent conformation, probably assisting the formation of the core.

Acyl-CoA Dehydrogenase↗