Intersubunit interactions in Plasmodium falciparum thioredoxin reductase

The thioredoxin redox system is composed of the NADPH-dependent homodimeric flavoprotein thioredoxin reductase (TrxR) and the 12-kDa protein thioredox in, It is responsible for the reduction of disulfide bridges in proteins su ch as ribonucleotide reductase and several transcription factors. Furthermo re, thioredoxin is involved in the detoxification of hydrogen peroxide and protects the cell against oxidative damage. There exist two classes of TrxR s: the high M-r and the low IM,proteins, The well characterized Escherichia coli TrxR represents a member of the low IM, class of proteins, whereas th e mammalian, Caenorhabditis elegans, and Plasmodium falciparum proteins bel ong to the family of high M-r proteins. The primary structure of these prot eins is very similar to that of glutathione reductase and lipoamide dehydro genase. However, the high M-r TrxRs possess, in addition to their redox act ive N-terminal pair of cysteines, a pair of cysteine residues or a seleneny lsulfide; motif at their C terminus. These residues have been shown to be c rucial for the reduction of thioredoxin, In this study we address the quest ion whether the active site residues of P, falciparum TrxR are provided by one or both subunits. Differentially tagged wild-type and PfTrxR mutants we re co-expressed in E, coli and the recombinant protein species were purifie d by affinity chromatography specific for the respective tags of the recomb inant proteins. Co-expression of PfTrxR wild-type and mutant proteins resul ted in the formation of three different protein species: homodimeric PfTrxR wild-type proteins, homodimeric mutant proteins, and heterodimers composed of one PfTrxR wild-type subunit and one PfTrxR mutant subunit. Go-expressi on of the double mutant PfTrxRC88AC535A with PfTxR wild-type generated an i nactive heterodimer, which indicates that PfTrxR possesses intersubunit act ive sites. In addition, the data presented possibly imply a coopertive inte raction between both active sites of PfTrxR.