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Fig. 2. EPR spectra of various ISCs from the electron transport chain. (Curve a) N-1a + N-1b [31], (curve b) S-1 [32], (curve c) Rieske [33] and (curve d) the sum with equal weight of N-1a, N-1b and S-1 clusters. (Curve e) reference spectrum of DNIC with thiol ligands.

stoichiometry of ISCs in electron transport chains in animal mitochondria [32]. We explicitly assumed that the relative contributions of the various ISCs in macrophages are similar to that in the mitochondrial chains of animal tissues.

Quantification of the spin density in these tissues poses a technical challenge since the EPR spectra appear as a superposition of broad signals from the various ISCs in the electron transport chain. We used the following protocol to determine the ratio of iron in ISPs vs. DNIC with EPR in the temperature range 40-80 K. First, we measured the double integrals Sdnic and 5isc of DNIC and sum of N-1a + N-1b + S-1, respectively. The absorption peaks are indicated in Fig. 2 as X (for DNIC) or Z (for ISC).

The small contribution from Rieske ISP was considered negligible. The ratio 5Dnic/5isc is proportional to X/Z, and the proportionality factor was calibrated by numerical simulation of the experimental EPR spectra as a mixture of DNIC and a synthetic ISC spectrum. The synthetic spectrum was generated as the equimolar mixture of experimental EPR signals of N-1a, N-1b and S-1 ISC, described in Refs. [31-33]. Technical details of the procedure are given in Ref. [24]. From the numerical calculations [24], we found that the two paramagnetic species are related as 5isc/5dnic = 5.0 ± 0.1 when X/Z = 1. Having in mind that the ISPs in 5isc are binuclear clusters [19,30-32] we find an iron ratio of [Feisc]/[FeDNic] = 10. In other words, we find that the paramagnetic population 5isc (N-1a + N-1b + S-1)

contains 10-fold more iron than the DNICs when XjZ = 1 [24]. However, these ratios only account for the EPR visible subfraction of iron in the electron transport chain, and the total iron content of the chain is significantly higher still. It was reported [19,30-33] that the full electron transport chain in animal mitochondria contains a total of 28 iron atoms in ICSs: 16 {[Fe2S2] N-1a, [Fe2S2]N-1b, [Fe4S4] N-2, [Fe4S4] N-3, [Fe4S4] N-4}, 8 {[Fe2S2]S-1, [Fe2S2]S-2, [Fe4S4]S-3} and 4 {[Fe2S2]-1 and [Fe2S2]-Rieske ISC} in the 1st, 2nd and 3rd Green's complexes, respectively. Therefore, the paramagnetic population 5isc (N-1a + N-1b + S-1) represents ca 20% of total "iron-sulfur" iron [total FeIsc] in the transport chain. Therefore, the total iron ratio is found [24] as

In this relation, [FeoNic] represents the iron sequestered in the form of the DNIC. All of this iron is EPR visible. The [total Feisc] represents the cumulative iron from all ISC of the electron transport chain, and only 20% of this iron is included in 5Isc.

After exposure to NO, prominent DNIC signal was visible in the preparation pre-treated with dithionite-methyl viologen (Fig. 1, curve g). At 40 K and 50 mW microwave power, the ratio of X/Z was equal to ~10 (Fig. 1, curves g,h). However, under these conditions, the DNIC signal at g = 2.03 is compressed by significant power saturation. The unsaturated X/Z ratio should be higher. Fig. 3 illustrates the effect of power saturation on the EPR intensity of DNIC in macrophage preparations.

As followed from the figure, at 60 K the EPR intensity is independent of microwave power if the latter exceeds 3 mW. Such behavior is characteristic of inhomogenous saturation of the DNIC complexes [34,35]. It was shown earlier [35] that the EPR signal from the DNIC begins to saturate namely at 3 mW of microwave power when the signal is recorded at higher temperature (77 K). So it is reasonable to suggest that the signal begins to saturate at microwave power of ca 2 mW when recorded at 40 K (Fig. 1, curve g). Therefore, at this g = 2.03 2.00

Fig. 3. EPR spectra of macrophage preparations treated with dithionite-methyl viologen and by aqueous NO solution (10-7 M, 5 min exposure). Spectra were taken at 60 K with microwave power of 50 mW (curve a) and 3 mW (curve b). (From Ref. [24].)

3.0 nnT B

value of microwave power (in the absence of saturation) the amplitude of the EPR signal of DNIC should be larger by a factor of ca (50/2)1/2 = 5 than that at 5 mW microwave power saturation. It means that at low microwave powers below saturation, the value of X/Z estimated from Fig. 1, curves g,h (~10) increase by a factor of (50/2)1/2 to ca 50!

This high X/Z ratio shows that within experimental error of ca 10%, the quantity of iron in DNIC was equal to the total iron in ISCs. It proves that the mechanism of DNIC formation cannot be simple disruption of the ISC clusters by free NO molecules, since there was no notable change in EPR signal intensities for ISCs treated with NO solution (Fig. 1, curves d-f and g-i, respectively). It is likely that iron sequestered in DNIC mainly originated from sources other than the tight-binding ISC. As discussed in Chapter 2, loosely bound iron from the so-called labile iron pool (LIP) [36] can contribute to the formation of DNIC (see Chapter 2).

The existence of such loosely bound "non-heme non-FeS" iron in rat liver mitochondria was demonstrated by optical spectroscopy in Ref. [37]. The optical assay was based on the formation of a chelate of Fe2+ with bathophenanthroline sulfonate (BPS) in osmotically swollen mitochondria. Fig. 4 shows that the quantity of paramagnetic ISCs was not affected by the addition of BPS and shows that the clusters did not lose their iron to this particular chelator. The EPR signal from the ISC was lost under the action of BPS only when the mitochondria were exposed to sodium dodecyl sulfate and acid pH.

The quantity of "non-heme non-FeS iron" was estimated from the optical density of the Fe-PBS complexes. Its value was nearly double the quantity of iron in ISCs as estimated from EPR (3.1 ± 0.6 nM Fe-PBS/mg protein vs. 1.7 ± 0.3 nM Fe-ISC/mg protein). The major part of BPS-chelated iron was confined to the mitochondrial inner compartment, i.e. matrix and inner membrane. More than half of the "non-heme non-FeS iron" of the "inner" pool was in the ferrous state in mitochondria as isolated. The biological significance of this iron

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