J

Fig. 5. EPR spectra of perfused mouse liver recorded at 77 K (curves a,b) or 20 K (curves c,d). (Curves b,d) initial preparations; (curves a,c) preparations after exposure to gaseous NO at the pressure of 200 mmHg for 30 min at ambient temperature. The amplifications of spectra (curves c,d) are equal to 1.0 and 1.5, respectively. (From Ref. [23].)

Fig. 5. EPR spectra of perfused mouse liver recorded at 77 K (curves a,b) or 20 K (curves c,d). (Curves b,d) initial preparations; (curves a,c) preparations after exposure to gaseous NO at the pressure of 200 mmHg for 30 min at ambient temperature. The amplifications of spectra (curves c,d) are equal to 1.0 and 1.5, respectively. (From Ref. [23].)

pool remained obscure. This pool was discussed as the source of iron for the synthesis of heme by ferrochelatase. It is reasonable to propose that this pool of "non-heme non-FeS iron" in mitochondria provides a significant fraction of the iron needed for the formation of some DNIC upon exposure to free NO or NO donors. This proposition would explain the formation of DNIC in preparations of rat liver mitochondria when treated with nitrite [38].

Fig. 5 shows independent experiments on perfused mouse liver preparations [23]. In this case, the presence of endogenous substrates was sufficiently high to bring all ISPs to reduced state without the addition of exogenous reductants. EPR showed that the ISCs together contained ca 1.5-2.0 mg Fe/g wet tissue, in reasonable agreement with the value of 1.3-1.5 mg/g wet tissues from the literature [19,30,31]. Upon exposure to gaseous NO, the ISPs were not noticeably degraded but EPR showed the formation of DNIC containing 1-1.5 mg Fe/g of wet tissue. The result shows that significant DNIC was formed in the liver tissue without concomitant loss of ISCs.

The same situation applies to the formation of DNIC from endogenous NO in liver tissue from mice. These animals were injected with bacteria C. parvum and the endogenous NO production was stimulated by injection with bacterial lipopolysaccharide [39] (Fig. 6).

Similar results were obtained with liver tissue from mice injected with bacteria C. sporogenus treated with nitrite as NO donors [25]. The above body of data attests to a remarkable stability of the ISC in the respiratory chain against disruption induced by NO treatment.

j 80 Gauss

Fig. 6. EPR spectra measured at 77 K of mouse liver. (Curve a) a normal mouse; (curve b) seven days after injection of 1 mg C. parvum (the liver was obtained 2 h after saline injection); (curve c) seven days after injection of 1 mg C. parvum (the liver was obtained 2 h after 1 Mg LPS injection) and (curve d) 2 h after injection of 300 Mg LPS. (From Ref. [39].)

j 80 Gauss

Fig. 6. EPR spectra measured at 77 K of mouse liver. (Curve a) a normal mouse; (curve b) seven days after injection of 1 mg C. parvum (the liver was obtained 2 h after saline injection); (curve c) seven days after injection of 1 mg C. parvum (the liver was obtained 2 h after 1 Mg LPS injection) and (curve d) 2 h after injection of 300 Mg LPS. (From Ref. [39].)

However, this stability is not a general rule since certain other ISCs show much higher sensitivity to such type of disruption. For example, Escherichia coli contain [Fe2S2] SoxR protein, a transcription factor modulating the expression of soxRS gene. In intact E. coli, 2-min exposure to gaseous NO led to the formation of the DNICs together with a strong decrease of the EPR signal of reduced SoxR protein. The loss of signal was attributed to the degradation of its active [Fe2S2] ICS. The same sensitivity to NO was seen in isolated SoxR protein [15]. Other ISPs like aconitase, ferrochelatase, endonuclease III or Chromatium vinosum high-potential iron protein have shown a clear and quantitative correlation between formation of DNIC and loss of ISC induced by exposure to NO [9,12,14,40]. For these cases, the experimental results are compatible with a mechanism as shown in Scheme 1. Details are discussed in Chapter 6. The above body of data can be summarized by noting that various ISCs show widely different susceptibility to degradation induced by NO exposure. Certain ISCs are very sensitive and easily disrupted by the formation of DNIC. For these ISCs, the disruption is plausibly explained by the mechanism of Scheme 1. The ISCs from the respiratory chain are very different in that they are robust against even high levels of NO. For these respiratory ISCs, the reaction shown in Scheme 1 seems irrelevant. Thus, the question arises which mechanism can degrade the ISC in the proteins of the mitochondrial chain?

Fig. 7. Changes of EPR spectra of reduced adrenodoxin (Adr) induced by nitrite treatment: (curve a) initial Adr preparation reduced by dithionite, (curve b) Adr treated by freezing-thawing in the presence of 2 mM guanidine-HCl followed by the addition of nitrite + dithionite under anaerobic conditions. (curve c) sample (b) after air exposure. Receiver gain for (a) is two times as high as that for (b) and (c). Recordings were made at 77 K [42].

As a model, we investigated the reaction between NO and [Fe2S2] ISC of Adr isolated from bovine adrenal glands. In vertebrates, Adr is a well-characterized [Fe2S2] ferredoxin. Adr mediates the electron transfer from NADPH via the Adr-reductase flavoenzyme to the cytochrome P450 of the respirational chain [41]. Adr has a [Fe2S2] ICS as presented in Scheme 1. In the reduced state, the [Fe2S2] cluster of Adr contains a ferric and a ferrous iron with antiferromagnetic coupling of the electronic spins to yield a total spin S = 1/2. This reduced binuclear cluster is easily observed with EPR and appears usually as an axially symmetric spectrum with main intensity at g± = 1.938 and g = 2.022 [18,19] (Fig. 7, top).

Our first experiments date back to the 1970s, where reduced 0.2 mM Adr [42] demonstrated that the spectral shape and intensity at g = 1.94 remained unaffected by repeated cycles of freezing-thawing and by the addition of the NO, producing a mixture of nitrite and dithionite. These experiments showed directly that NO alone did not induce significant loss of ICS in Adr (Fig. 7). Similarly, the combination of nitrite (0.25 mM) and dithionite failed to affect the EPR spectrum of reduced Adr after pre-incubation with protein denaturant guanidine-HCl (0.4 mM) at anaerobic conditions for 3-5 min. However, loss of ISC could be induced by freezing-thawing the solution with reduced Adr and guanidine-HCl prior to the addition of nitrite + dithionite. Under anaerobic conditions, EPR showed the formation of a mixture of two types of paramagnetic DNIC with {3d7} and {3d9} configurations (Fig. 7). These two redox states are easily distinguished from differences in line position and lineshape (cf Chapter 2). The EPR absorption by the ISC of Adr at g = 1.94 disappeared completely. Upon exposure to ambient air, all {3d9} DNICs were oxidized to {3d7} and only the 2.03 signal remained (Fig. 7). Double integration of the EPR spectrum showed that the total spin density from both DNIC species accounted for ca 80% of the spin density of the binuclear

1.94 signal. Therefore, the total iron content of the DNIC amounts to ca 40% of the iron originally in the [Fe2S2] clusters of Adr.

Thus, the formation of DNIC in these experiments was only initiated after degradation of this center by another mechanism. We propose that Adr is degraded according to Scheme 2. The mechanism involves loss of the bridging inorganic sulfur atoms (S*) from the iron. Throughout the degradation the iron remains bound to protein thiol groups and the subsequent DNIC appears protein bound as well.

"S

denaturation

Was this article helpful?

0 0

Post a comment