I

Magnetic Field (mT)

Fig. 2. Effect of biological thiols on protein DNIC. A ferredoxin-DNIC sample (10 /u,M; trace a) was incubated with no addition (trace b) or with 1 mM l-cysteine (Cys; trace c), 1 mM N-acetyl-l-cysteine (NAC; trace d) or 1 mM glutathione (GSH; trace e) at 37°C for 20min, followed by EPR spectroscopy. Reproduced from Rogers and Ding [53].

alone, the amplitude of the EPR signal at gav = 2.04 of the sample was not affected (Fig. 2, trace b). However, when the ferredoxin DNIC was incubated with L-cysteine, the gav = 2.04 EPR signal was completely eliminated (Fig. 2, trace c). Oxygen in solution had little effect, as L-cysteine decomposed the protein-bound DNIC under both aerobic and anaerobic conditions [53].

D-cysteine had the same effect as L-cysteine (data not shown), which indicates that the redox properties of cysteine rather than its stereo configuration are critical for decomposing the ferredoxin DNIC. However, the monothiols N-acetyl-L-cysteine and glutathione had no effect (Fig. 2, traces d,e), which was also true of reduced thioredoxin (not shown). Thus, cysteine seems to have unique activity in eliminating the DNIC EPR signal and perhaps the protein-bound DNICs themselves.

Fig. 3. Correlation of iron release and EPR intensity during incubation of ferredoxin DNIC with l-cysteine at 37°C. Reproduced from Rogers and Ding [53].

l-CYSTEINE releases ferrous iron from the protein-bound

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