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Fig. 9. EPR spectra from 0.2 mM adrenodoxin (Adr) treated with 56Fe2+ (1.8 mM) + NO (curve a) followed by thiosulfate (curves b,c). Recordings were made at 77 K, microwave power 20 mW and modulation amplitude 0.5 mT (curves a,b) and 0.01 mT (curve c). To the right of the spectra, relative amplifications of the signals are shown. Reproduced with permission, from Ref. [21] © the Biochemical Society.

thiosulfate was added to the solution, a narrow isotropic signal at g = 2.03 appeared that was accompanied by a decline in the EPR signal from protein-bound DNIC (Fig. 9).

This was due to the transfer of Fe+(NO+)2 moieties from the latter to thiosulfate ions, resulting in the formation of thiosulfate-DNIC of low molecular weight. The mobility of these complexes at ambient temperature was high enough to average the aforementioned anisotropy. Reducing the amplitude of the field modulation from 0.5 to 0.01 mT led to the appearance of a quintet HFS originated from two 14N atoms in NO+ ligands (see Chapter 2).

Similar results were obtained in the experiments with Adr in oxidized state [2]. The contact of this protein (0.2 mM) with gaseous NO (at the pressure of 100 mmHg) did not result in the degradation of ISC in Adr. Subsequent reduction with dithionite showed the presence of the same quantity of intact ISC at g = 1.94 as in preparations not exposed to NO (Fig. 10). The experimental spectra show some residual broad EPR absorption in the g-factor range 2.07-1.98 from heme-nitrosyl complexes as well as a small EPR signal from DNIC. The intensities of these signals did not exceed 10 and 1 ^M, respectively. All indicates that the ISC of Adr is very robust towards NO alone.

The situation changes completely in the presence of exogenous iron: The DNIC formation in this preparation increased the amount of the complexes of 0.2-0.3 mM when 1.8 mM ferrous iron was added to the solution prior to the NO treatment (Fig. 10). Concomitantly, all

Fig. 10. EPR spectra at 77 K from 0.2 mM oxidized adrenodoxin (Adr) treated with dithionite, NO + dithionite, Fe2+ (1.8 mM) + NO, or Fe2+ (1.8 mM) + NO + dithionite. Amplification factors are shown on the right side. (From Ref. [22].)

Fig. 11. EPR spectra at room temperature from 0.2 mM oxidized adrenodoxin (Adr) treated with FeSOz Amplification factors are shown on the right side. (From Ref. [22].)

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Fig. 11. EPR spectra at room temperature from 0.2 mM oxidized adrenodoxin (Adr) treated with FeSOz Amplification factors are shown on the right side. (From Ref. [22].)

ISCs in Adr were destroyed. The DNIC was protein bound, because the EPR lineshape remained unchanged when the temperature was raised from 77 K to ambient (Fig. 11, curve a). The treatment of protein-bound DNIC with dithionite resulted in a sharp decrease of the EPR signal of {3d7}-DNIC and new EPR absorption with g± = 2.01 and g = 1.97 from {3d9}-DNIC (Fig. 11, curve b). The effect of dithionite treatment was reversible: exposure to air restored the signal from {3d7}-DNIC (Fig. 11, curve c). However, experiments at room temperature show that the population of {3d7}-DNIC was a mixture of protein-bound DNIC and low-weight DNIC. The registration of the narrow symmetric EPR signal at g = 2.03 overlapping the anisotropic signal from protein-bound DNIC was due to the formation of low-molecular DNIC with thiosulfate ligands originating from the dithionite.

The strong effect of ferrous iron could be explained by the formation of low-molecular DNICs from the exogenous iron and NO with subsequent action of these DNICs on the [Fe2S2] ICS of Adr. Our experiments suggest that the degradation of ICSs proceed via a mechanism involving a DNIC of low molecular weight (Scheme 3).

The reaction consumes two low-molecular DNICs and a single ISC and results in the formation of two protein-bound DNICs and the release of endogenous *Fe iron and inorganic

Scheme 3. Degradation of reduced ISC induced by the action of DNIC with low-molecular weight [21]. L- is a small anionic ligand, for example thiosulfate or cysteine.

sulfur S* atoms. At sufficient levels of L- and NO, the released iron can again form new low-molecular DNICs, and attack the next ISC. In this way, the DNIC of low molecular weight acts as a catalyst for the denaturation of the ISC in Adr. Fig. 10 suggests that Scheme 3 is applicable to oxidized Adr as well. Possibly, it could apply to tetranuclear [Fe4S4] clusters also.

The validity of Scheme 3 was supported by many experimental observations: First, in the absence of NO gas, the addition of 1.8 mM phosphate-DNIC to reduced Adr (0.2 mM) led to full degradation of ISC as observed at g = 1.94. EPR demonstrated the complete transfer of the DNIC moiety from low to high molecular weight. Second, the addition of 0.3 mM 57 Fe (nuclear spin I = 1/2) instead of abundant 56Fe (I = 0) to reduced Adr solution in the presence of NO led to a DNIC spectrum with significant isotopic broadening of the gx component due to HFS from 57Fe (for details of this isotopic broadening cf Chapter 2). With 2.1 mT, the width at half amplitude (5) of the gx component of the 57Fe-DNIC is more than that of 56Fe-DNIC with 1.6 mT (Figs. 8 and 11).

The linewidth proves that the DNIC had predominantly formed from the exogenous iron rather than the endogenous unlabeled iron in the ISC. The isotopic ratio could be shifted by subsequent exposure to gaseous NO: 56Fe-DNIC from the endogenous iron pool began to dominate when the preparation was subsequently treated with NO and dithionite. This was clearly reflected in the sharpening of the DNIC spectrum at g = 2.03 (Fig. 12). The change in lineshape proves that the mechanism of DNIC formation and ISC degradation involves a mixing of the endogenous (57Fe) and exogenous (56Fe) iron pools. The increase of the latter in the DNIC composition can point to auto-catalytical mechanism of DNIC formation and ISC degradation.

The relative contributions of exogenous (57Fe) and endogenous (56Fe) iron were investigated by adding an excess quantity of thiosulfate (5 mM) to the preparation resulting in the formation of low-molecular DNIC with thiosulfate (Fig. 9). The kinetics of the process could be monitored with EPR on a timescale of minutes at ambient temperature. The EPR spectra of thiosulfate-DNIC are sensitive to the isotopic labeling with 57Fe. At low amplitude of the field modulation (0.01 mT), the isotropic DNIC-thiosulfate spectrum has resolved quintet HFS with nitrogens of the nitrosyl ligands and additional doublet HFS from 57Fe (I = 1/2) (Fig. 13). As expected, the EPR spectra of mixtures of 57Fe and 56Fe isotopes show a complicated HFS pattern (Fig. 13).

A similar complicated HFS pattern was recorded for the solution of pre-reduced Adr (0.2 mM) following the addition of NO and an equimolar mixture of 57Fe- and 56Fe-citrate (0.2 mM), with 5 mM thiosulfate added 5 min later (Fig. 14, curve a). The intensity of the EPR signal increased and the HFS pattern changed when this preparation was additively treated with NO and dithionite (Fig. 14, curve b, right side). The spectrum could be well simulated with a 1.5:1 mixture of 56Fe-DNIC and 57Fe-DNIC (Fig. 14, curve b, left side).

The enrichment of DNIC with 56Fe proves that endogenous iron from ISC began to make contribution to the formation of DNIC-thiosulfate. This conclusion is supported by exposing reduced Adr with NO and exogenous 57Fe (0.3 mM) without 56Fe. It results in the formation of thiosulfate-ligated DNIC with isotopic ratio 56Fe:57Fe = 0.44:1 (Fig. 14, curve c). Subsequent exposure to gaseous NO and dithionite increased the ratio to 0.75:1 (Fig. 14, curve d).

The above data can be summarized as follows: The degradation of the [Fe2S2] cluster in Adr and formation of DNIC cannot be achieved by free NO molecules alone. In particular, the mechanism of Scheme 1 is not valid for Adr. In vitro, we found that this ferredoxin was very susceptible to degradation by the action of low-molecular DNICs independently from reduced or oxidized state of ISC Adr. This action is rapid and significant already at low DNIC concentrations. Such small quantities of DNIC will form spontaneously in any solution containing small anionic ligands, NO and spurious quantities of free or loosely bound iron. Thiols are known to be particularly effective ligands for the formation of DNIC. We note that all three ingredients are readily available in actual biological systems. Therefore, it seems plausible that the attack of low-molecular DNIC on binuclear ISC be relevant for in vivo conditions as well. We propose that the attack of low-molecular DNIC on [Fe2S2] of Adr proceed according to Scheme 3. This reaction mechanism forms apo-ISP-bound DNIC.

We have seen above that certain classes of proteins do not show functional correlation between ISC degradation and formation of DNIC. In particular, breakup of the ISC was

Fig. 12. EPR spectra at 77 K from 0.2 mM reduced adrenodoxin (Adr) (curve a); addition of 57Fe2+ (0.3 mM) + NO to the preparation (a) (curve b); addition of dithionite to preparation (b) (curve c). Amplification factors are shown on the right. S is the width of the signal at half amplitude. Reproduced with permission, from Ref. [21] © the Biochemical Society.

not initiated by NO exposure alone. For these NO-resistant proteins, the degradation was achieved by the action of low-molecular DNIC. It is not to be excluded that certain ISCs are resistant to small DNIC as well. For example, geometrical constraints may leave ISC inaccessible to low-molecular DNIC. The latter are small but certainly bulky in comparison with the highly mobile NO radical. Blocked access could potentially protect the active centers of ISPs buried in lipid compartments, for example in the mitochondrial electron transport chain.

It seems tempting to attribute all protein-bound DNIC in cells and tissues as originating from the degradation of ISCs via Scheme 3. However, in vitro studies have shown that low-molecular DNIC may readily transfer their Fe(NO+ )2 moiety to other thiol groups on the protein, i.e. to thiols not at all involved in the ISC. We are convinced that a significant fraction of protein-bound DNIC is anchored to such non-ISC thiols. It is even conceivable that the majority of protein-bound DNICs be anchored on non-ISC thiols. The presence of various cellular compartments with different polarity and dielectric properties complicates the in vivo situation even further. Although the elucidation of these problems needs further investigations, it is already clear that Scheme 1 cannot be taken as a paradigm for the degradation of all types of ISCs, and that the ISC of Adr and the respiratory chain form a different class. In vitro experiments with Adr have demonstrated that the presence of small DNIC complexes promotes catalytic breakup of ISC under concomitant formation of DNIC, as well as exchange between the iron pools of ISC an DNIC. The efficiency of this catalytic reaction suggests that it may have physiological relevance for biological systems.

Fig. 13. EPR spectra from the solutions of DNIC with thiosulfate containing 57Fe (upper trace), 56Fe (middle trace) or an equimolar mixture of 57Fe and 56Fe (lower trace). Recordings were made at ambient temperature, microwave power 20 mW and modulation amplitude 0.01 mT. Reproduced with permission, from Ref. [21] © the Biochemical Society.

Fig. 13. EPR spectra from the solutions of DNIC with thiosulfate containing 57Fe (upper trace), 56Fe (middle trace) or an equimolar mixture of 57Fe and 56Fe (lower trace). Recordings were made at ambient temperature, microwave power 20 mW and modulation amplitude 0.01 mT. Reproduced with permission, from Ref. [21] © the Biochemical Society.

fitted spectra g = 2.03

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