Figure 2.

Fdx-dependent reduction of GvTrx-m and GvCP12 via GvDTR. A, Schematic illustration of the in vitro reaction performed in this study for the reduction of Trx (B), and further GvCP12 (C), by GvDTR with electrons derived from [2Fe-2S] GvFdx, itself reduced by Anabaena’s FNR in the presence of NADPH. B, The reaction mixtures contained equal amounts of Trx, NADPH, and some or all of the components included in the scheme depicted in (A), as indicated. After reaction, free sulfhydryl groups were labeled with AMS, which increases the mass of the protein by about 0.5 KDa per free thiol, allowing the two different redox states of GvTrx-m (reduced or oxidized, Trx_red or Trx_ox respectively) to be separated by SDS-PAGE under nonreducing conditions. GvTrx-m in the oxidized state (buffer) and in the reduced state (after DTT incubation) are included for comparison. C, In a similar kind of reaction as above, the reduced/oxidized ratio of GvCP12 was determined by non-reducing SDS-PAGE after AMS treatment in the presence of components according to the complete scheme indicated in (A). The images of the complete gels can be found in Supplemental Figures S2–S3.