The riboflavin/FAD cycle in rat liver mitochondria

Here we provide evidence that mitochondria isolated from rat liver can synt hesize FAD from riboflavin that has been taken up and from endogenous ATP. Riboflavin uptake takes place via a carrier-mediated process, as shown by t he inverse relationship between fold accumulation and riboflavin concentrat ion, the saturation kinetics [riboflavin K-m and V-max values were 4.4 +/- 1.3 mu m and 35 +/- 5 pmol.min(-1).(mg protein)(-1), respectively] and the inhibition shown by the thiol reagent mersalyl, which cannot enter the mito chondria. 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 A TP and FMN synthesized from taken up riboflavin via the putative mitochondr ial riboflavin kinase. In the light of certain features, including the prot ein thermal stability and molecular mass, mitochondrial FAD synthetase diff ers from the cytosolic isoenzyme. Apparent K-m and apparent V-max values fo r FMN were 5.4 +/- 0.9 mu m and 22.9 +/- 1.4 pmol.min(-1).(mg matrix protei n)(-1), respectively. Newly synthesized FAD inside the mitochondria can be exported from the mitochondria in a manner sensitive to atractyloside but i nsensitive to mersalyl. The occurrence of the riboflavin/FAD cycle is propo sed to account for riboflavin uptake in mitochondria biogenesis and ribofla vin recovery in mitochondrial flavoprotein degradation; both are prerequisi tes for the synthesis of mitochondrial flavin cofactors.