redox-sensitive Cys residues at the active sites of the NTR and the TRX domains are
essential for activity (Perez-Ruiz and Cejudo 2009). Based on these data it was
proposed that the catalytically active form of NTRC is a homodimer arranged in a
head-to-tail conformation. The enzyme shows much higher affinity for NADPH than
for NADH. Thus, NADPH is the source of reducing power, which is transferred to
FAD and then to the double Cys at the active site of the NTR domain of one of the
subunits. There is then an inter-subunit transfer of electrons to the active site double
Cys of the TRX domain of the other subunit of the enzyme, which interacts with the
disulfide of the 2-Cys PRX (Perez-Ruiz and Cejudo 2009). According to this model,
stating that NTRC interacts with its targets by the TRX domain, it could be
considered that NTRC is a TRX with its own reductase, which might be the reason
of the high catalytic efficiency of this enzyme (Cejudo et al. 2012). The high affinity
of NTRC for NADPH and the proposed electron transfer pathway was further
confirmed by stopped-flow spectroscopy (Bernal-Bayard et al. 2012), and the
interaction between NTRC and 2-Cys PRX was also demonstrated in vivo by
bimolecular fluorescence complementation analyses (Bernal-Bayard et al. 2014).
In line with this notion it was found that NTRC is unable to interact with other
plastidial TRX, hence, having no activity as NTR (Bohrer et al. 2012). However, the
Arabidopsis ntrc mutant, which is devoid of NTRC, was partially complemented by
the expression of a mutated version of the NTRC gene encoding an enzyme with
intact NTR and inactive TRX domains, indicating that the NTR domain of NTRC
might interact with other plastidial TRX among which TRXf was proposed as the
most likely partner (Toivola et al. 2013). Therefore, whether NTRC acts exclusively
as a TRX with its reductase incorporated or is able to interact with other plastidial
TRX, hence showing NTR activity, still requires further analysis.
Finally, it should be mentioned that NTRC shows ability to oligomerize in vitro.
While for the enzyme from rice this ability is redox sensitive, the enzyme being
detected as dimer in the presence of NADPH and as oligomer in its absence (PerezRuiz et al. 2006), the oligomerization capacity of the enzyme from barley is redox
insensitive (Wulff et al. 2011). The oligomeric form of NTRC shows chaperone and
holdase activity, which might be the reason of the tolerance to elevated temperature
of plants overexpressing NTRC (Chae et al. 2013).
2.2.3 NTRC Controls the Redox State of 2-Cys PRX
Based on the fact that NTRC is an efficient reductant of 2-Cys PRX, hence
supporting the hydrogen peroxide scavenging activity of these enzymes, it was
proposed that NTRC has antioxidant activity (Perez-Ruiz et al. 2006). However, it
was also shown that different chloroplast TRX are able to reduce 2-Cys PRX, the
most efficient one being TRXx (Collin et al. 2003). Thus, a relevant issue was to
establish the hierarchy of the different chloroplast redox systems for the reduction of
2-Cys PRX. The redox state of the 2-Cys PRX in vivo, determined as the ratio of
dimeric (oxidized) to monomeric (reduced) forms of the enzyme, was severely
impaired in an Arabidopsis mutant devoid of NTRC, which contained almost no
234
I. Thormählen et al.
essential for activity (Perez-Ruiz and Cejudo 2009). Based on these data it was
proposed that the catalytically active form of NTRC is a homodimer arranged in a
head-to-tail conformation. The enzyme shows much higher affinity for NADPH than
for NADH. Thus, NADPH is the source of reducing power, which is transferred to
FAD and then to the double Cys at the active site of the NTR domain of one of the
subunits. There is then an inter-subunit transfer of electrons to the active site double
Cys of the TRX domain of the other subunit of the enzyme, which interacts with the
disulfide of the 2-Cys PRX (Perez-Ruiz and Cejudo 2009). According to this model,
stating that NTRC interacts with its targets by the TRX domain, it could be
considered that NTRC is a TRX with its own reductase, which might be the reason
of the high catalytic efficiency of this enzyme (Cejudo et al. 2012). The high affinity
of NTRC for NADPH and the proposed electron transfer pathway was further
confirmed by stopped-flow spectroscopy (Bernal-Bayard et al. 2012), and the
interaction between NTRC and 2-Cys PRX was also demonstrated in vivo by
bimolecular fluorescence complementation analyses (Bernal-Bayard et al. 2014).
In line with this notion it was found that NTRC is unable to interact with other
plastidial TRX, hence, having no activity as NTR (Bohrer et al. 2012). However, the
Arabidopsis ntrc mutant, which is devoid of NTRC, was partially complemented by
the expression of a mutated version of the NTRC gene encoding an enzyme with
intact NTR and inactive TRX domains, indicating that the NTR domain of NTRC
might interact with other plastidial TRX among which TRXf was proposed as the
most likely partner (Toivola et al. 2013). Therefore, whether NTRC acts exclusively
as a TRX with its reductase incorporated or is able to interact with other plastidial
TRX, hence showing NTR activity, still requires further analysis.
Finally, it should be mentioned that NTRC shows ability to oligomerize in vitro.
While for the enzyme from rice this ability is redox sensitive, the enzyme being
detected as dimer in the presence of NADPH and as oligomer in its absence (PerezRuiz et al. 2006), the oligomerization capacity of the enzyme from barley is redox
insensitive (Wulff et al. 2011). The oligomeric form of NTRC shows chaperone and
holdase activity, which might be the reason of the tolerance to elevated temperature
of plants overexpressing NTRC (Chae et al. 2013).
2.2.3 NTRC Controls the Redox State of 2-Cys PRX
Based on the fact that NTRC is an efficient reductant of 2-Cys PRX, hence
supporting the hydrogen peroxide scavenging activity of these enzymes, it was
proposed that NTRC has antioxidant activity (Perez-Ruiz et al. 2006). However, it
was also shown that different chloroplast TRX are able to reduce 2-Cys PRX, the
most efficient one being TRXx (Collin et al. 2003). Thus, a relevant issue was to
establish the hierarchy of the different chloroplast redox systems for the reduction of
2-Cys PRX. The redox state of the 2-Cys PRX in vivo, determined as the ratio of
dimeric (oxidized) to monomeric (reduced) forms of the enzyme, was severely
impaired in an Arabidopsis mutant devoid of NTRC, which contained almost no
234
I. Thormählen et al.