Electron transfer pathways and dynamics of chloroplast NADPH-dependent thioredoxin reductase C (NTRC)

Abstract

NADPH-dependent thioredoxin reductases (NTRs) contain a flavin cofactor and a disulfide as redox-active groups. The catalytic mechanism of standard NTR involves a large conformational change between two configurations. Oxygenic photosynthetic organisms possess a plastid-localized NTR, called NTRC, with a thioredoxin module fused at the C terminus. NTRC is an efficient reductant of 2-Cys peroxiredoxins (2-Cys Prxs) and thus is involved in the protection against oxidative stress, among other functions. Although the mechanism of electron transfer of canonical NTRs is well established, it is not yet known in NTRC. By employing stopped-flow spectroscopy, we have carried out a comparative kinetic study of the electron transfer reactions involving NTRC, the truncated NTR module of NTRC, and NTRB, a canonical plant NTR. Whereas the three NTRs maintain the conformational change associated with the reductive cycle of catalysis, NTRC intramolecular electron transfer to the thioredoxin module presents two kinetic components (k(ET) of ~2 and 0.1 s(-1)), indicating the occurrence of additional dynamic motions. Moreover, the dynamic features associated with the electron transfer to the thioredoxin module are altered in the presence of 2-Cys Prx. NTRC shows structural constraints that may locate the thioredoxin module in positions with different efficiencies for electron transfer, the presence of 2-Cys Prx shifting the conformational equilibrium of the thioredoxin module to a specific position, which is not the most efficient.

FIGURE 1.

Representative spectra for the progressive reduction of 20 μm NTRB (upper panel) and NTRC (lower panel) samples by adding repeated fixed amounts (1 μm) of NADPH; top spectra, initial spectra without NADPH; bottom spectra, plus 45 μm (upper panel) or 65 μm (lower panel) NADPH. Other experimental conditions were as described under “Experimental Procedures.”

FIGURE 2.

Upper panel, transient kinetics of NTRC reduction by NAD(P)H were observed by stopped flow at 456 nm; kinetic traces could be adjusted to multiexponential fits, as indicated under “Results.” NTRC- and NAD(P)H-concentrated samples were mixed to obtain final concentrations of 7.5 μm NTRC and 27 μm NADPH or 270 μm NADH. Upper panel inset, NTRC reduction by NADPH measured at 540 nm. Lower panel, dependence on NADPH concentration of the observed rate constants (k_obs) of the intermediate phase for NTRC reduction. Lower panel inset, dependence on NADH concentration of k_obs for NTRC reduction. Experiments were carried out in 20 mm phosphate buffer, pH 7.5, at 10 °C under anaerobic conditions. NTRC reduction was followed by mixing 200-μl solutions of NTRC 8 μm with small volumes of concentrated nucleotide solutions (0.3–3 mm). Continuous lines represent theoretical fits according to the reaction mechanisms previously proposed (39). Other experimental conditions were as described under “Experimental Procedures.”

FIGURE 3.

Upper panel, transient kinetics for NTRC intramolecular electron transfer, after reduction by NADPH, as observed by stopped flow at 456 nm in the absence or presence of 2-Cys Prx or Trx x; kinetic traces of reoxidation could be adjusted to multiexponential fits, as indicated under “Results.” NTRC and NADPH concentrated samples were mixed to obtain final concentrations of 15 μm NTRC and 15 μm NADPH, in the absence or the presence of 50 μm 2-Cys Prx or Trx x, as indicated. For comparative purposes, a similar experiment was carried out with NTRC_M. Lower panel, dependence on NADPH concentration of the k_obs for the two reoxidation phases. NTRC reactions were followed by mixing 200-μl solutions of NTRC 15 μm with small volumes of concentrated NADPH solutions (0.09–0.3 mm). Other experimental conditions were as described in Fig. 2.

FIGURE 4.

Transient kinetics for the reoxidation reactions in NTRC_Cys-1 (A) or NTRC_Cys-2 (B) samples and in NTRC_Cys-1/NTRC_Cys-2 1:1 mixtures (C). Inset, reoxidation reaction in NTRC_Cys-1/NTRC_Cys-2 1:1 mixtures in the presence of 50 μm 2-Cys Prx. Intramolecular electron transfer in the NTRC mutants, after reduction by NADPH, was followed by stopped flow at 456 nm. NTRC mutants and NADPH concentrated samples were mixed to obtain final concentrations of 20 μm protein and 20 μm NADPH (A and B) or 30 μm protein and 30 μm NADPH (C, and inset). Other experimental conditions were as described in Fig. 3.

FIGURE 5.

Proposed model for the reaction mechanism of NTRC. In NTRC dimers, the minimal catalytic unit, electron transfer from NTR to Trx modules occurs via inter-subunit pathways. In the absence of 2-Cys Prx (upper panel), the equilibrium between different structural conformations of the Trx domain related to the NTR module in each monomer (indicated as dashed gray arrows) would establish different electron transfer rates. 2-Cys Prx shifts the equilibrium restricting the Trx module to a specific configuration (lower panel). Each monomer in NTRC dimers is colored in dark or light gray, respectively; white arrows indicate electron transfer pathways.