Mitochondrial Redox Metabolism in Trypanosomatids Is Independent of Tryparedoxin Activity Helena Castro1., Susana Romao1., Sandra Carvalho1, Filipa Teixeira1, Carla Sousa1, Ana M. Toma´ s1,2* 1 IBMC - Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal, 2 ICBAS - Instituto de Cieˆncias Biome´dicas Abel Salazar, Universidade do Porto, Porto, Portugal Abstract Tryparedoxins (TXNs) are oxidoreductases unique to trypanosomatids (including Leishmania and Trypanosoma parasites) that transfer reducing equivalents from trypanothione, the major thiol in these organisms, to sulfur-dependent peroxidases and other dithiol proteins. The existence of a TXN within the mitochondrion of trypanosomatids, capable of driving crucial redox pathways, is considered a requisite for normal parasite metabolism. Here this concept is shown not to apply to Leishmania. First, removal of the Leishmania infantum mitochondrial TXN (LiTXN2) by gene-targeting, had no significant effect on parasite survival, even in the context of an animal infection. Second, evidence is presented that no other TXN is capable of replacing LiTXN2. In fact, although a candidate substitute for LiTXN2 (LiTXN3) was found in the genome of L. infantum, this was shown in biochemical assays to be poorly reduced by trypanothione and to be unable to reduce sulfur- containing peroxidases. Definitive conclusion that LiTXN3 cannot directly reduce proteins located within inner mitochondrial compartments was provided by analysis of its subcellular localization and membrane topology, which revealed that LiTXN3 is a tail-anchored (TA) mitochondrial outer membrane protein presenting, as characteristic of TA proteins, its N-terminal end (containing the redox-active domain) exposed to the cytosol. This manuscript further proposes the separation of trypanosomatid TXN sequences into two classes and this is supported by phylogenetic analysis: i) class I, encoding active TXNs, and ii) class II, coding for TA proteins unlikely to function as TXNs. Trypanosoma possess only two TXNs, one belonging to class I (which is cytosolic) and the other to class II. Thus, as demonstrated for Leishmania, the mitochondrial redox metabolism in Trypanosoma may also be independent of TXN activity. The major implication of these findings is that mitochondrial functions previously thought to depend on the provision of electrons by a TXN enzyme must proceed differently. Citation: Castro H, Romao S, Carvalho S, Teixeira F, Sousa C, et al. (2010) Mitochondrial Redox Metabolism in Trypanosomatids Is Independent of Tryparedoxin Activity. PLoS ONE 5(9): e12607. doi:10.1371/journal.pone.0012607 Editor: Pedro Lagerblad Oliveira, Universidade Federal do Rio de Janeiro, Brazil Received June 22, 2010; Accepted August 13, 2010; Published September 8, 2010 Copyright: 2010 Castro et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by grants PPCDT/SAU-IMI/59560/2004 and PTDC/CVT/70275/2006 from Fundac¸a˜o para a Cieˆncia e a Tecnologia (http://alfa. fct.mctes.pt/) and P-105335 from Fundac¸a˜o Calouste Gulbenkian (http://www.gulbenkian.pt/). HC, SR and SC are recipients of FCT fellowships (SFRH/BPD/20610/ 2004, SFRH/BD/18452/2004 and SFRH/BD/28712/2006, respectively). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: atomas@ibmc.up.pt . These authors contributed equally to this work. Introduction Tryparedoxins (or TXNs) are oxidoreductases found exclusively in organisms of the family Trypanosomatidae, which includes medically relevant protozoan parasites of the genera Leishmania and Trypanosoma. TXNs are remotely related to enzymes of the thioredoxin family. When compared to these, TXNs share only 13% sequence identity, are larger in size (molecular weight of 16 kDa versus 11 kDa for thioredoxins) and exhibit a unique active site signature that reads Trp-Cys-Pro-Pro-Cys-Arg [instead of Trp–Cys–(Gly/Ala)–Pro–Cys–Lys for most thioredoxins]. Moreover, unlike thioredoxins, which are reduced enzymatically by thioredoxin reductase, TXNs are specifically reduced by trypanothione [T(SH)2], a conjugate of glutathione and spermi- dine that largely replaces glutathione functions in trypanosomatids and that is itself maintained in a reduced state by the flavoenzyme trypanothione reductase at the expense of NADPH. The reducing equivalents obtained from trypanothione are transferred by TXNs to a variety of thiol-containing molecules [1]. Accordingly, as thioredoxins, TXNs participate in different aspects of parasite development, many of which are crucial to life. The first characterized activity of TXNs was the reduction of 2- Cys peroxiredoxins (2-Cys PRXs) [2], a ubiquitous class of sulfur- dependent peroxidases. For this reason, trypanosomatid 2-Cys PRXs were coined as tryparedoxin peroxidases or TXNPxs. The subsequent observation that TXNs are also the reductants for non- selenium glutathione peroxidase-like enzymes (nsGPXs) [3,4,5] reinforced the idea that TXNs are central players in trypanoso- matid peroxide detoxification. Other TXN oxidants include ribonucleotide reductase [6], the universal minicircle sequence binding protein (UMSBP) [7] and a monothiol glutaredoxin (1- Cys GRX) [8,9], i.e. molecules that are required for nuclear and mitochondrial DNA replication, and for iron-sulfur cluster biosynthesis, respectively. TXNs can be found in the cytosol of all trypanosomatids, as is the case for the TXN1 molecules of Leishmania infantum [10], Trypanosoma brucei [11], Trypanosoma cruzi [3] and Crithidia fasciculata [12]. The importance of cytosolic TXNs for survival has been PLoS ONE | www.plosone.org 1 September 2010 | Volume 5 | Issue 9 | e12607