Trypanosoma brucei and Trypanosoma cruzi Tryparedoxin Peroxidases Catalytically Detoxify Peroxynitrite via Oxidation of Fast Reacting Thiols

Abstract

Macrophage activation is one of the hallmarks observed in trypanosomiasis, and the parasites must cope with the resulting oxidative burden, which includes the production of peroxynitrite, an unusual peroxo-acid that acts as a strong oxidant and trypanocidal molecule. Cytosolic tryparedoxin peroxidase (cTXNPx) has been recently identified as essential for oxidative defense in trypanosomatids. This peroxiredoxin decomposes peroxides using tryparedoxin (TXN) as electron donor, which in turn is reduced by dihydrotrypanothione. In this work, we studied the kinetics of the reaction of peroxynitrite with the different thiol-containing components of the cytosolic tryparedoxin peroxidase system in T. brucei (Tb) and T. cruzi (Tc), namely trypanothione, TXN, and cTXNPx. We found that whereas peroxynitrite reacted with dihydrotrypanothione and TbTXN at moderate rates (7200 and 3500 m–1 s–1, respectively, at pH 7.4 and 37 °C) and within the of the for the reaction of peroxynitrite with reduced and and at pH 7.4 and 37 This a identified as that as the of that and is by TXN, that a for peroxynitrite in peroxynitrite in Macrophage activation is one of the hallmarks observed in trypanosomiasis, and the parasites must cope with the resulting oxidative burden, which includes the production of peroxynitrite, an unusual peroxo-acid that acts as a strong oxidant and trypanocidal molecule. Cytosolic tryparedoxin peroxidase (cTXNPx) has been recently identified as essential for oxidative defense in trypanosomatids. This peroxiredoxin decomposes peroxides using tryparedoxin (TXN) as electron donor, which in turn is reduced by dihydrotrypanothione. In this work, we studied the kinetics of the reaction of peroxynitrite with the different thiol-containing components of the cytosolic tryparedoxin peroxidase system in T. brucei (Tb) and T. cruzi (Tc), namely trypanothione, TXN, and cTXNPx. We found that whereas peroxynitrite reacted with dihydrotrypanothione and TbTXN at moderate rates (7200 and 3500 m–1 s–1, respectively, at pH 7.4 and 37 °C) and within the of the for the reaction of peroxynitrite with reduced and and at pH 7.4 and 37 This a identified as that as the of that and is by TXN, that a for peroxynitrite in peroxynitrite in brucei and cruzi the of and respectively, of in and by an in the of and the of activation in T. brucei is an whereas T. cruzi the and in the of T. and T. of which has in and in In as and as a of the oxidative by T. and T. by the the of the in the of by by of reaction and the of peroxynitrite and a strong and T. cruzi in the system of with T. brucei brucei and in the of and which has been peroxynitrite peroxynitrite is peroxynitrite and and peroxynitrite is peroxynitrite and and is an unusual has a of the in and a of that an that decomposes by in pH 7.4 and 37 °C) and which with of and reaction with in and in and with and for peroxynitrite in that by reaction and by the with and is a that the of and the in the of an a resulting in an of peroxynitrite reaction and the of the for the and at pH 7.4 the is an of that with peroxynitrite at rates of at pH 7.4 and 37 the peroxiredoxin peroxynitrite has been that in at the of which of the as as the of is an the of in is by a of that the dihydrotrypanothione the of reduced and of the dihydrotrypanothione the reduced and the of trypanothione, the of reduced and of the dihydrotrypanothione the reduced and the of trypanothione, for the of in and T. Cytosolic tryparedoxin peroxidase cytosolic tryparedoxin T. brucei TXN, T. brucei T. cruzi cytosolic tryparedoxin T. brucei TXN, T. brucei T. cruzi which is a is essential for in T. brucei and for and by is T. cruzi of by in tryparedoxin of and an a that with of an an of has been for of peroxynitrite this of oxidant has been recently in the of In this has been that whereas with peroxynitrite with tryparedoxin in the as the (TXN) as an electron and and is reduced by which in turn is by a and a at the of In T. peroxynitrite dihydrotrypanothione the and has been that the system peroxynitrite the of within the oxidative of the of dihydrotrypanothione and peroxynitrite at the and tryparedoxin peroxidase thiol-containing in with the of as of peroxynitrite in in by and we the of different components of the tryparedoxin peroxidase system in namely trypanothione, and with peroxynitrite, with the the of peroxynitrite in T. brucei and T. and by with and of T. brucei and T. brucei and T. cruzi Cytosolic by of the in of of T. brucei as identified by as an that as of TbTXN T. brucei brucei cytosolic and of the as as T. cruzi cytosolic as in with the T. cruzi the and in at with in and of with the in a with and with pH at a of in pH in a and and as a of peroxynitrite with the in of peroxynitrite at pH of peroxynitrite of with and pH in pH 7.4 and with and in the of reduced by of reduced using the and and reduced by the of a of by a with a at and in pH which for with at by the as as by at as T. of by by of the with a of for as as using the of in with and at at kinetics of peroxynitrite studied in a with a of peroxynitrite is at m–1 we at m–1 in by at the the peroxynitrite at a rates of peroxynitrite the by the of peroxynitrite at and by the at peroxynitrite which peroxynitrite at pH In for peroxynitrite by with the of at at 37 and the pH at the by peroxynitrite at pH 7.4 and 37 °C) the in a that at the as the of at In the of the for reaction with peroxynitrite as at pH 7.4 and 37 of of by T. at 37 and the pH at the of in the of by the for the reaction of the and peroxynitrite at pH 7.4 and 37 as the In this peroxynitrite and with the reduced with the kinetics of the reaction studied using a and the of of the the for peroxynitrite as the of at peroxynitrite that reduced the of the using the and at different as with the and using of with and T. brucei of dihydrotrypanothione peroxynitrite an of the of peroxynitrite at pH 7.4 and 37 reacted with peroxynitrite with an of m–1 at pH 7.4 and 37 In of the of peroxynitrite that the of the the for the for the reaction of peroxynitrite and different thiol-containing in T. brucei and T. pH 7.4 and 37 pH 7.4 and 37 in a reduced T. brucei tryparedoxin a peroxynitrite s–1, that the peroxynitrite and the reduced is m–1 at pH 7.4 and 37 of the of T. brucei Cytosolic with the of peroxynitrite an an of at pH 7.4 and 37 in peroxynitrite rates by of reduced with as for the of peroxynitrite with the in the and at of with In the for the reaction and peroxynitrite, we using in which by with peroxynitrite, with an of s–1, whereas a the of peroxynitrite and a of at pH 7.4 and 37 rates of peroxynitrite reaction with and peroxynitrite rates in pH and 37 with by of peroxynitrite peroxynitrite reduced in pH 7.4 and 37 in the of by the peroxynitrite a that by we peroxynitrite in the of in the in the of reduced that is reduced by reduced TbTXN with a of at pH and peroxynitrite the the as that the for the of by by we a of TbTXN that reduced in the of peroxynitrite has been peroxynitrite with reduced TbTXN a in peroxynitrite whereas the of peroxynitrite reduced a peroxynitrite by a of peroxynitrite that of the in the of reduced and reduced TbTXN peroxynitrite an that at the of the of the of peroxynitrite at the of an for the reaction peroxynitrite and reduced of at pH 7.4 and 37 This is the by the for the In TbTXN and TbTXN a peroxynitrite in the of reduced is in the of TbTXN and in pH 7.4 and 37 at a of the of peroxynitrite of the of T. cruzi Cytosolic with peroxynitrite by reduced by the a of for the reaction peroxynitrite and reduced at pH 7.4 and 37 in the of a system by reduced and reduced TbTXN an that by the for the reaction peroxynitrite and with the in s–1, at pH 7.4 and 37 with the by the decomposes peroxynitrite in the of in the of of and reduced TbTXN in pH 7.4 and 37 at and of of of peroxynitrite reduced an of the of the that peroxynitrite the of of the in with the of the reaction for of of peroxynitrite in the in the of of in the of of by reaction peroxynitrite and reduced that the by of which and and we in We as a and with peroxynitrite and the of reduced of the of and the of peroxynitrite an of the peroxynitrite and the as is an that is reduced the of with and of the the of and at observed for the of peroxynitrite is the of and one the reaction of peroxynitrite with which is the in and the in the reaction of peroxynitrite with in a for the reaction peroxynitrite and of at pH 7.4 and 37 in with the and a for the reaction of peroxynitrite with of at pH 7.4 and 37 which is the for the reaction of by peroxynitrite in pH 7.4 and 37 at by the in the of of and the using using a for the reaction peroxynitrite and the of of by peroxynitrite as in the of the in the of the in the of the which the observed for peroxynitrite of in reaction of and which at a with peroxynitrite with and has been and peroxynitrite in of a of peroxynitrite at pH 7.4 and which a of of reduced in which the the of in the of reduced TbTXN and reduced the with reduced reduced TbTXN that by of in pH 7.4 and with in the of reduced and in the of and with of the thiol-containing components of the T. brucei cytosolic tryparedoxin peroxidase system TXN, and and the for the reaction of peroxynitrite and reduced is with dihydrotrypanothione reduced TbTXN at identified as the peroxynitrite in the as is for the reaction with and for has been activation by a of that is the of and by which that in tryparedoxin is at with a which that in this the is essential for whereas the is essential for peroxynitrite the and and the of this is the reaction of peroxynitrite with of the of peroxynitrite reduced the of of the of the reaction by peroxynitrite with the for which a of the an in the of of the of and which of the the reaction with and and a in the of an a the of a the of at of peroxynitrite with and the with of peroxynitrite that with is the of peroxynitrite in the of the by the of peroxynitrite in the of m–1 and the observed in in the of is the the and in of and which this an for peroxynitrite the peroxynitrite in the the in is that the of the the of the with and the of the by in a that for This the TbTXN with peroxynitrite at the in a peroxynitrite the of and TbTXN an in the of peroxynitrite that the of the TbTXN and peroxynitrite is a that is reduced by TbTXN acts as a by a of peroxynitrite, the of in the of and TbTXN in the of the of using that this decomposes peroxynitrite in a and the for the reaction the and peroxynitrite an and of of peroxynitrite by the of T. and peroxynitrite the for the the In the of T. which is an peroxynitrite is in the is T. brucei by the in in the of the which is a T. with and peroxynitrite the of T. brucei and T. cruzi as an for the and of in the of brucei and cruzi the of and respectively, of in and by an in the of and the of activation in T. brucei is an whereas T. cruzi the and in the of T. and T. of which has in and in In as and as a of the oxidative by T. and T. by the the of the in the of by by of reaction and the of peroxynitrite and a strong and T. cruzi in the system of with T. brucei brucei and in the of and which has been peroxynitrite peroxynitrite is peroxynitrite and and peroxynitrite is peroxynitrite and and is an unusual has a of the in and a of that an that decomposes by in pH 7.4 and 37 °C) and which with of and reaction with in and in and with and for peroxynitrite in that by reaction and by the with and is a that the of and the in the of an a resulting in an of peroxynitrite reaction and the of the for the and at pH 7.4 the is an of that with peroxynitrite at rates of at pH 7.4 and 37 the peroxiredoxin peroxynitrite has been that in at the of which of the as as the of is an the of in is by a of that the dihydrotrypanothione the of reduced and of the dihydrotrypanothione the reduced and the of trypanothione, the of reduced and of the dihydrotrypanothione the reduced and the of trypanothione, for the of in and T. Cytosolic tryparedoxin peroxidase cytosolic tryparedoxin T. brucei TXN, T. brucei T. cruzi cytosolic tryparedoxin T. brucei TXN, T. brucei T. cruzi which is a is essential for in T. brucei and for and by is T. cruzi of by in tryparedoxin of and an a that with of an an of has been for of peroxynitrite this of oxidant has been recently in the of In this has been that whereas with peroxynitrite with Cytosolic tryparedoxin in the as the (TXN) as an electron and and is reduced by which in turn is by a and a at the of In T. peroxynitrite dihydrotrypanothione the and has been that the system peroxynitrite the of within the oxidative of the of dihydrotrypanothione and peroxynitrite at the and tryparedoxin peroxidase thiol-containing in with the of as of peroxynitrite in in by and we the of different components of the tryparedoxin peroxidase system in namely trypanothione, and with peroxynitrite, with the the of peroxynitrite in T. brucei and T. and by with and of T. brucei and T. brucei and T. cruzi Cytosolic by of the in of of T. brucei as identified by as an that as of TbTXN T. brucei brucei cytosolic and of the as as T. cruzi cytosolic as in with the T. cruzi the and in at with in and of with the in a with and with pH at a of in pH in a and and as a of peroxynitrite with the in of peroxynitrite at pH of peroxynitrite of with and pH in pH 7.4 and with and in the of reduced by of reduced using the and and reduced by the of a of by a with a at and in pH which for with at by the as as by at as T. of by by of the with a of for as as using the of in with and at at kinetics of peroxynitrite studied in a with a of peroxynitrite is at m–1 we at m–1 in by at the the peroxynitrite at a rates of peroxynitrite the by the of peroxynitrite at and by the at peroxynitrite which peroxynitrite at pH In for peroxynitrite by with the of at at 37 and the pH at the by peroxynitrite at pH 7.4 and 37 °C) the in a that at the as the of at In the of the for reaction with peroxynitrite as at pH 7.4 and 37 of of by T. at 37 and the pH at the of in the of by the for the reaction of the and peroxynitrite at pH 7.4 and 37 as the In this peroxynitrite and with the reduced with the kinetics of the reaction studied using a and the of of the the for peroxynitrite as the of at peroxynitrite that reduced the of the using the and at different as with the and using and by with and of T. brucei and T. brucei and T. cruzi Cytosolic by of the in of of T. brucei as identified by as an that as of TbTXN T. brucei brucei cytosolic and of the as as T. cruzi cytosolic as in with the T. cruzi the and in at with in and of with the in a with and with pH at a of in pH in a and and as a of peroxynitrite with the in of peroxynitrite at pH of peroxynitrite of with and pH in pH 7.4 and with and in the of reduced by of reduced using the and and reduced by the of a of by a with a at and in pH which for with at by the as as by at as T. of by by of the with a of for as as using the of in with and at at kinetics of peroxynitrite studied in a with a of peroxynitrite is at m–1 we at m–1 in by at the the peroxynitrite at a rates of peroxynitrite the by the of peroxynitrite at and by the at peroxynitrite which peroxynitrite at pH In for peroxynitrite by with the of at at 37 and the pH at the by peroxynitrite at pH 7.4 and 37 °C) the in a that at the as the of at In the of the for reaction with peroxynitrite as at pH 7.4 and 37 of of by T. at 37 and the pH at the of in the of by the for the reaction of the and peroxynitrite at pH 7.4 and 37 as the In this peroxynitrite and with the reduced with the kinetics of the reaction studied using a and the of of the the for peroxynitrite as the of at peroxynitrite that reduced the of the using the and at different as with the and using of with and T. brucei of dihydrotrypanothione peroxynitrite an of the of peroxynitrite at pH 7.4 and 37 reacted with peroxynitrite with an of m–1 at pH 7.4 and 37 In of the of peroxynitrite that the of the the for the for the reaction of peroxynitrite and different thiol-containing in T. brucei and T. pH 7.4 and 37 pH 7.4 and 37 in a reduced T. brucei tryparedoxin a peroxynitrite s–1, that the peroxynitrite and the reduced is m–1 at pH 7.4 and 37 of the of T. brucei Cytosolic with the of peroxynitrite an an of at pH 7.4 and 37 in peroxynitrite rates by of reduced with as for the of peroxynitrite with the in the and at of with In the for the reaction and peroxynitrite, we using in which by with peroxynitrite, with an of s–1, whereas a the of peroxynitrite and a of at pH 7.4 and 37 by of peroxynitrite peroxynitrite reduced in pH 7.4 and 37 in the of by the peroxynitrite a that by we peroxynitrite in the of in the in the of reduced that is reduced by reduced TbTXN with a of at pH and peroxynitrite the the as that the for the of by by we a of TbTXN that reduced in the of peroxynitrite has been peroxynitrite with reduced TbTXN a in peroxynitrite whereas the of peroxynitrite reduced a peroxynitrite by a of peroxynitrite that of the in the of reduced and reduced TbTXN peroxynitrite an that at the of the of the of peroxynitrite at the of an for the reaction peroxynitrite and reduced of at pH 7.4 and 37 This is the by the for the In TbTXN and TbTXN a peroxynitrite in the of reduced is in the of TbTXN and in pH 7.4 and 37 at a of the of peroxynitrite of the of T. cruzi Cytosolic with peroxynitrite by reduced by the a of for the reaction peroxynitrite and reduced at pH 7.4 and 37 in the of a system by reduced and reduced TbTXN an that by the for the reaction peroxynitrite and with the in s–1, at pH 7.4 and 37 with the by the decomposes peroxynitrite in the of in the of of and reduced TbTXN in pH 7.4 and 37 at and of of of peroxynitrite reduced an of the of the that peroxynitrite the of of the in with the of the reaction for of of peroxynitrite in the in the of of in the of of by reaction peroxynitrite and reduced that the by of which and and we in We as a and with peroxynitrite and the of reduced of the of and the of peroxynitrite an of the peroxynitrite and the as is an that is reduced the of with and of the the of and at observed for the of peroxynitrite is the of and one the reaction of peroxynitrite with which is the in and the in the reaction of peroxynitrite with in a for the reaction peroxynitrite and of at pH 7.4 and 37 in with the and a for the reaction of peroxynitrite with of at pH 7.4 and 37 which is the for the reaction of by peroxynitrite in pH 7.4 and 37 at by the in the of of and the using using a for the reaction peroxynitrite and the of of by peroxynitrite as in the of the in the of the in the of the which the observed for peroxynitrite of in reaction of and which at a with peroxynitrite with and has been and peroxynitrite in of a of peroxynitrite at pH 7.4 and which a of of reduced in which the the of in the of reduced TbTXN and reduced the with reduced reduced TbTXN that by of in pH 7.4 and with in the of reduced and in the of and of with and T. brucei of dihydrotrypanothione peroxynitrite an of the of peroxynitrite at pH 7.4 and 37 reacted with peroxynitrite with an of m–1 at pH 7.4 and 37 In of the of peroxynitrite that the of the the for the reduced T. brucei tryparedoxin a peroxynitrite s–1, that the peroxynitrite and the reduced is m–1 at pH 7.4 and 37 of the of T. brucei Cytosolic with the of peroxynitrite an an of at pH 7.4 and 37 in peroxynitrite rates by of reduced with as for the of peroxynitrite with the in the and at of with In the for the reaction and peroxynitrite, we using in which by with peroxynitrite, with an of s–1, whereas a the of peroxynitrite and a of at pH 7.4 and 37 peroxynitrite a that by we peroxynitrite in the of in the in the of reduced that is reduced by reduced TbTXN with a of at pH and peroxynitrite the the as that the for the of by by we a of TbTXN that reduced in the of peroxynitrite has been peroxynitrite with reduced TbTXN a in peroxynitrite whereas the of peroxynitrite reduced a peroxynitrite by a of peroxynitrite that of the in the of reduced and reduced TbTXN peroxynitrite an that at the of the of the of peroxynitrite at the of an for the reaction peroxynitrite and reduced of at pH 7.4 and 37 This is the by the for the In TbTXN and TbTXN a peroxynitrite of the of T. cruzi Cytosolic with peroxynitrite by reduced by the a of for the reaction peroxynitrite and reduced at pH 7.4 and 37 in the of a system by reduced and reduced TbTXN an that by the for the reaction peroxynitrite and with the in s–1, at pH 7.4 and 37 with the by the of of of peroxynitrite reduced an of the of the that peroxynitrite the of of the in with the of the reaction for of of peroxynitrite in the in the of of in the of of by reaction peroxynitrite and reduced that the by of which and and we in We as a and with peroxynitrite and the of reduced of the of and the of peroxynitrite an of the peroxynitrite and the as is an that is reduced the of with and of the the of and at observed for the of peroxynitrite is the of and one the reaction of peroxynitrite with which is the in and the in the reaction of peroxynitrite with in a for the reaction peroxynitrite and of at pH 7.4 and 37 in with the and a for the reaction of peroxynitrite with of at pH 7.4 and 37 which is the for the reaction of in reaction of and which at a with peroxynitrite with and has been and peroxynitrite in of a of peroxynitrite at pH 7.4 and which a of of reduced in which the the of in the of reduced TbTXN and reduced the with reduced reduced TbTXN that by with of the thiol-containing components of the T. brucei cytosolic tryparedoxin peroxidase system TXN, and and the for the reaction of peroxynitrite and reduced is with dihydrotrypanothione reduced TbTXN at identified as the peroxynitrite in the as is for the reaction with and for has been activation by a of that is the of and by which that in tryparedoxin is at with a which that in this the is essential for whereas the is essential for peroxynitrite the and and the of this is the reaction of peroxynitrite with of the of peroxynitrite reduced the of of the of the reaction by peroxynitrite with the for which a of the an in the of of the of and which of the the reaction with and and a in the of an a the of a the of at of peroxynitrite with and the with of peroxynitrite that with is the of peroxynitrite in the of the by the of peroxynitrite in the of m–1 and the observed in in the of is the the and in of and which this an for peroxynitrite the peroxynitrite in the the in is that the of the the of the with and the of the by in a that for This the TbTXN with peroxynitrite at the in a peroxynitrite the of and TbTXN an in the of peroxynitrite that the of the TbTXN and peroxynitrite is a that is reduced by TbTXN acts as a by a of peroxynitrite, the of in the of and TbTXN in the of the of using that this decomposes peroxynitrite in a and the for the reaction the and peroxynitrite an and the of T. and peroxynitrite the for the the In the of T. which is an peroxynitrite is in the is T. brucei by the in in the of the which is a T. with and peroxynitrite the of T. brucei and T. cruzi as an for the and of in the of with of the thiol-containing components of the T. brucei cytosolic tryparedoxin peroxidase system TXN, and and the for the reaction of peroxynitrite and reduced is with dihydrotrypanothione reduced TbTXN at identified as the peroxynitrite in the as is for the reaction with and for has been activation by a of that is the of and by which that in tryparedoxin is at with a which that in this the is essential for whereas the is essential for peroxynitrite the and and the of this is the reaction of peroxynitrite with of the of peroxynitrite reduced the of of the of the reaction by peroxynitrite with the for which a of the an in the of of the of and which of the the reaction with and and a in the of an a the of a the of at of peroxynitrite with and the with of peroxynitrite that with is the of peroxynitrite in the of the by the of peroxynitrite in the of m–1 and the observed in in the of is the the and in of and which this an for peroxynitrite the peroxynitrite in the the in is that the of the the of the with and the of the by in a that for This the TbTXN with peroxynitrite at the in a peroxynitrite the of and TbTXN an in the of peroxynitrite that the of the TbTXN and peroxynitrite is a that is reduced by TbTXN acts as a by a of peroxynitrite, the of in the of and TbTXN in the of the of using that this decomposes peroxynitrite in a and the for the reaction the and peroxynitrite an and In the of T. and peroxynitrite the for the the In the of T. which is an peroxynitrite is in the is T. brucei by the in in the of the which is a T. with and peroxynitrite the of T. brucei and T. cruzi as an for the and of in the of We of for and for and with with