The riboflavin/FAD cycle in rat liver mitochondria

Abstract

Here we provide evidence that mitochondria isolated from rat liver can synthesize FAD from riboflavin that has been taken up and from endogenous ATP. Riboflavin uptake takes place via a carrier-mediated process, as shown by the inverse relationship between fold accumulation and riboflavin concentration, the saturation kinetics [riboflavin K m and V max values were 4.4^1.3 mm and 35^5 pmol´min 21´( mg protein) 21 , respectively] and the inhibition shown by the thiol reagent mersalyl, which cannot enter the mitochondria. FAD synthesis is due to the existence of FAD synthetase (EC 2.7.7.2), localized in the matrix, which has as a substrate pair mitochondrial ATP and FMN synthesized from taken up riboflavin via the putative mitochondrial riboflavin kinase. In the light of certain features, including the protein thermal stability and molecular mass, mitochondrial FAD synthetase differs from the cytosolic isoenzyme. Apparent K m and apparent V max values for FMN were 5.4^0.9 mm and 22.9^1.4 pmol´min 21´( mg matrix protein) 21 , respectively. Newly synthesized FAD inside the mitochondria can be exported from the mitochondria in a manner sensitive to atractyloside but insensitive to mersalyl. The occurrence of the riboflavin/FAD cycle is proposed to account for riboflavin uptake in mitochondria biogenesis and riboflavin recovery in mitochondrial flavoprotein degradation; both are prerequisites for the synthesis of mitochondrial flavin cofactors.Keywords: FAD synthetase; flavin cofactors; mitochondria; riboflavin; transport.Although the biochemistry of mammalian mitochondria has been investigated in depth, the mechanisms of uptake and/or processing of vitamins and/or vitamin-derived cofactors, often necessary for assembly with their apoenzymes already imported from the cytosol, remain to be fully elucidated.The uptake of certain vitamins by mitochondria has already been reported to occur via different mechanisms, including diffusion followed by binding to intramitochondrial protein(s) [1], simple diffusion [2±4] and carrier-mediated transport [5,6]. Mitochondria are able to synthesize certain cofactors from taken up precursors, namely adenosylcobalamin [7] and CoA [8]. In contrast, mitochondria can take up cytosolically synthesized cofactors. Diffusion followed by binding to a specific high-affinity intramitochondrial protein has been proposed for pyridoxal 5 H -phosphate uptake [2], whereas thiamine diphosphate (TPP) and CoA can enter the mitochondria via a carrier-mediated process [9±11]. NAD synthesis from externally added nicotinamide mononucleotide has been reported in rat liver mitochondria (RLM) [12], while NAD permeation in mitochondria has also been shown in human cells [13].The above-described state of the art requires investigation regarding the existence of mitochondrial translocators and enzymes involved in vitamin and/or vitamin cofactor transport and metabolism in mitochondria. In particular, because RLM contain a huge amount of FAD and FMN, elucidation of the processes by which mitochondria provide and re...

Flavinylation of the precursor of mitochondrial dimethylglycine dehydrogenase by intact and solubilised mitochondria

“…In both rat liver mitochondria (RLM) and Saccharomyces cerevisiae mitochondria, FAD synthesis occurs from ribo£avin and/or FMN due to the existence of a ribo£avin kinase (EC 2.7.1.26) and a FAD synthetase (EC 2.7.7.2). The latter has been characterised from RLM and localised in the mitochondrial matrix [5]. Consistently, ribo£avin kinase, the product of the FMN1 gene in S. cerevisiae, has been found to be localised in the inner mitochondrial membrane [6].…”

“…The mitochondrial matrix actively participates in holoMe 2 GlyDH biogenesis also by providing the £avin cofactor [5,9]. Considering that Me 2 GlyDH is synthesised in the cytosol as a precursor polypeptide, we asked whether mitochondrial component(s) are able to promote holoenzyme formation starting from the precursor.…”

Mitochondrial regulation of epigenetics and its role in human diseases

“…Bai et al analyzed mtDNA polymorphisms in EuropeanAmerican females and reported that A10398G and T16519C mitochondria involved in the regulation of these complex processes remains elusive. These collectively include SAM, 54 ATP (adenosine triphosphate) citrate lyase (ACL), Acetyl-CoA, 62 flavin adenine dinucleotide (FAD), 63 and lysine-specific demethylase 1 (LSD1). 64,65 Understanding the influence these factors have on epigenetic states of nuclear genomic DNA and signaling pathways could help identify roles bioenergetics, biogenesis and the mitochondria might have in regulating epigenetic modifications within the nuclear genome.…”

“…61 The level of cross talk between DNA methylation and chromatin state are beginning to be identified. An understanding of factors, co-factors and enzymes generated both outside and inside the nucleus and within the 65 The process of H3K4me2 demethylation catalyzed by LSD1 utilizes the classical amine oxidation reaction and FAD as a co-factor 63 ( Fig. 2A).…”

“…Both FAD and α-KG are known to be synthesized in mammalian mitochondria. 63,79,80 Reduced levels of these co-factors due to mitochondrial impairment/dysfunction could have significant effects on regulation of the nuclear genome. This could lead to perturbed demethylation of histone lysine residues catalyzed either by LSD1 or JMJD.…”