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Scheme 2. Proposed mechanism of DNIC formation in denaturated adrenodoxin (Adr) [42].

More recent experiments [21,22] on Adr were consistent with the earlier data [42]. The NO gas used in these latest experiments was purified by the method of low-temperature sublimation in an evacuated system [43] and good care was taken to remove even traces of the nitrogen dioxide radical (this oxidant species rapidly accumulates in NO gas, especially at higher pressures, cf Chapter 2). As shown in Fig. 8 [21], the addition of freshly prepared and purified gaseous NO to 0.2 mM pre-reduced solution of Adr resulted in the appearance of a weak DNIC absorption in the EPR spectrum of the solution. However, the amount of DNIC (2 ^M) calculated from the signal was much less than the loss of ca 40 ^M ISC as observed at g = 1.94.

The exposure to NO also led to the formation of ferrous heme-nitrosyl complexes from heme-protein admixture in Adr preparation as shown by the broad EPR signal in the g-factor range 2.07-1.98 at this treatment (Fig. 8). These results confirm that formation of DNIC and loss of ISC are quite independent processes in Adr.

The degradation of ISC and the formation of DNICs in the presence of NO was sharply accelerated by the addition of 1.8 mM Fe2+-citrate complex to the solutions (Fig. 8, recordings were made at 77 K). The yield of DNICs reached a maximum concentration of 0.3 ± 0.1 mM. This value was already reached at 2-3 min after the addition of iron, and was accompanied with practically complete degradation of the ISC of Adr. Subsequent treatment of the solution with dithionite resulted in a decrease of the intensity of the 2.03 signal and the formation of a new paramagnetic complex with gx = 2.01 and gy = 1.97 (Fig. 8). As was mentioned above, this signal was due to reduced form of DNICs with electron configuration {3d9} (cf Chapter 2). The reduction with dithionite did not restore the EPR signal from reduced Adr, thereby showing that the ISC had disintegrated rather than oxidized to diamagnetic state. No effect of Fe2+ -citrate complex on the ISC in Adr was observed.

When increasing the measurement temperature from 77 K to room temperature, the shape of the DNIC EPR signal did not change [21] (Fig. 9). This indicated unequivocally that the complexes remained bound to the protein globule: its low mobility was not sufficient to average the g-factor and hyperfine structure (HFS) anisotropy. However, when 5 mM

Fig. 8. EPR spectra from a 0.2 mM solution of adrenodoxin (Adr), treated with dithionite (curve a) followed with NO (curve b) or with 56Fe2+ (1.8 mM) + NO (curve c). (curve d) Treatment of preparation (c) with dithionite. Recordings were made at 77 K. To the right of the spectra, the relative amplifications of the signals are indicated. S is the width of the signal at half amplitude. Reproduced with permission, from Ref. [21] © the Biochemical Society.

Fig. 8. EPR spectra from a 0.2 mM solution of adrenodoxin (Adr), treated with dithionite (curve a) followed with NO (curve b) or with 56Fe2+ (1.8 mM) + NO (curve c). (curve d) Treatment of preparation (c) with dithionite. Recordings were made at 77 K. To the right of the spectra, the relative amplifications of the signals are indicated. S is the width of the signal at half amplitude. Reproduced with permission, from Ref. [21] © the Biochemical Society.

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