Xj

Rieske i_

Fig. 1. EPR spectra of control macrophage preparations [24]. Left panel: unreduced preparations at 25 K (curve a), 16 K (curve b) and 30 K (curve c). Right panel: preparations treated by dithionite (1 mM) + methyl viologen (5 mM), recorded at 40 K (curves d,e) and 23 K (curve f). Preparations treated with dithionite-methyl viologen after 5 min incubation with 10-7 M NO, recorded at 40 K (curves g,h) and 23 K (curve i). (Curve j) shows the EPR spectrum of mouse liver at 20 K.

absorption near g ~ 1.94 as recorded at 40 K was attributed to a superposition of signals from reduced [Fe2S2] centers like N-1b, N-1a, S-1 and Rieske ISCs [26,28,30]. Thus, the EPR spectra show that the ISC content of the respiratory chain in macrophages is similar to that in animal tissues. Further support for this similarity is seen in the EPR spectrum attributed to the various ISCs in mouse liver (Fig. 1, curve j).

Subsequently, we investigated the disruption by NO. The bolus injection of 100 nM NO to 107 macrophages in 0.3 ml buffer did not significantly affect the EPR spectra from reduced ISCs. In particular, there was no significant loss of intensity of their EPR signals. However, the EPR spectra showed the formation of a small quantity of paramagnetic DNICs centered at g = 2.03 (Fig. 1, curve g). It is noteworthy that NO induced the formation of significant quantities of DNIC only in macrophages treated with dithionite-methyl viologen. It did not matter whether the reduction was before or after NO addition. The EPR spectrum of DNIC is much narrower and sharper than that of reduced ISCs. Therefore, the derivative EPR spectra are much more sensitive to a gain in DNIC than for a loss of reduced ISC. In principle, double integration of the EPR spectrum should reveal the iron content in ISCs and DNICs, respectively. In practice, the strong EPR signal from methyl viologen radicals at g = 2.0 prevented the reliable quantification of the iron of the various ISCs. Therefore, the double integration was applied to a selection of the ISCs only, namely N-1a, N-1b and S-1. The EPR signals from these ISCs as well as the (small) Rieske [Fe2S2] were recorded at 40-80 K [24,31]. The amount of iron in the remaining ISCs was estimated from the known

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