Evidence for the presence of a FAD pyrophosphatase and a FMN phosphohydrolase in yeast mitochondria: a possible role in flavin homeostasis

Abstract

Despite the crucial roles of flavin cofactors in metabolism, we know little about the enzymes responsible for the turnover of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) and their subcellular localization. The mechanism by which mitochondria obtain their own flavin cofactors is an interesting point of investigation, because FMN and FAD are mainly located in mitochondria, where they act as redox cofactors of a number of dehydrogenases and oxidases that play a crucial function in both bioenergetics and cellular regulation. In this context, the capability of yeast mitochondria to metabolize externally added and endogenous FAD and FMN was investigated and use was made of purified and bioenergetically active mitochondria prepared starting from the Saccharomyces cerevisiae cell. To determine whether flavin metabolism can occur, the amounts of flavins in aliquots of neutralized perchloric extracts of both spheroplasts and mitochondria were measured by HPLC, and the competence of S. cerevisiae mitochondria to metabolize FAD and FMN was investigated both spectroscopically and via HPLC. FAD deadenylation and FMN dephosphorylation were studied with respect to dependence on substrate concentration, pH profile and inhibitor sensitivity. The existence of two novel mitochondrial FAD pyrophosphatase (diphosphatase) (EC 3.6.1.18) and FMN phosphohydrolase (EC 3.1.3.2) activities, which catalyse the reactions FAD + H₂O → FMN + AMP and FMN + H₂O → riboflavin + Pi respectively, is here shown by fractionation studies. Considering cytosolic riboflavin, FMN and FAD concentrations, as calculated by measuring both spheroplast and mitochondrial contents via HPLC, probably mitochondria play a major role in regulating the flavin pool in yeast and in relation to flavin homeostasis.