Research Article Evidence for the presence of a FAD pyrophosphatase and a FMN phosphohydrolase in yeast mitochondria: a possible role in flavin homeostasis Maria Luigia Pallotta * Department of Health Sciences, University of Molise, Campobasso, Italy *Correspondence to: M. L. Pallotta, Department of Health Sciences, University of Molise, Via F. De Sanctis, 86100 Campobasso, Italy. E-mail: pallotta@unimol.it Received: 8 March 2011 Accepted: 10 July 2011 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 mito- chondria 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 mito- chondria 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 + H 2 O ! FMN + AMP and FMN + H 2 O ! 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, prob- ably mitochondria play a major role in regulating the flavin pool in yeast and in relation to flavin homeostasis. Copyright © 2011 John Wiley & Sons, Ltd. Keywords: flavin homeostasis; Saccharomyces cerevisiae mitochondria; FAD pyropho- sphatase (diphosphatase); FMN phosphohydrolase; riboflavin Introduction The natural flavin cofactors flavin mononucleotide (FMN; riboflavin 5′-phosphate) and flavin adenine dinucleotide (FAD) differ from riboflavin (vitamin B 2 ) in their ribityl side chains, which contain a phosphate residue or an ADP moiety in ester link- age with its terminal hydroxyl group, respectively (Mansoorabadi et al., 2007). The tricyclic isoallo- sazine ring system is the reactive part of the flavin. It is an amphipathic molecule formed by the fusion of the hydrophobic dimethylbenzene moiety with the hydrophilic pyrimidine ring. FMN and FAD, the biologically active forms of riboflavin, play a lead role in a diverse array of biological processes, which is a reflection of their structural and chem- ical versatility (Joosten and van Berkel, 2007). At present, the Protein Data Base Bank (PDB) contains about 200 entries for FAD- and FMN- dependent proteins (Fraaije and Mattevi, 2000). Flavoenzymes have the unique ability to catalyse a wide range of biochemical reactions, as well as Yeast Yeast 2011; 28: 693–705. Published online 13 September 2011 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/yea.1897 Copyright © 2011 John Wiley & Sons, Ltd.