Info

RSNO [mol/mol of caspase 3] Caspase 3 activity [% Ctrl]

Fig. 2. Trxn denitrosates caspase 3. Experiments were carried out in 0.1 M phosphate buffer containing 0.1 mM desferrioxamine. S-nitrosation of caspase 3 (5 ^M) by GSNO (0.1 mM; A) and denitrosation by Trxn (10 ^M)/TrxnR (15 U/mL)/NADPH (0.2 mM; B) were carried out as described in Ref. [49]. Caspase activity was determined by using Enzcheck Caspase 3 assay kit (Molecular Probes, OR). Data are presented as mean values ± SE (n = 3).

these effects may re ect Trxn nitrosation in its redox inactive cysteine 69 to ONS-Cys(69)-Trxn-S2, which, in turn, inhibited activators of apoptosis by delivering NO to their active sites [47].

However, direct experiments with Trxn and caspases that support this mechanism have not been presented. This hypothesis was challenged by model experiments of Mitchell and Marletta who reported that GSNO preferentially nitrosates oxidized Trxn (Trxn-S2) in cysteine 73 without affecting cysteine 69. The authors proposed that ONS-Cys(73)-Trxn-S2 may act as an inhibitor of caspase 3 via trans- S-nitrosation of this protease to ONS-Cys(163) -caspase 3 [48]. Given these controversial data, it is important to elucidate whether (a) other nitrosating species (e.g. metal-nitrosyl complexes and S-nitroso(homo)cysteine) interact preferentially with Trxn-cysteine 69, and (b) ONS-Cys-Trxn-S2 undergoes auto-denitrosation to HS-Cys-Trxn-(SH)2 in physiologically relevant conditions that include the presence of TrxnR and NADPH. In our studies, the complete Trxn/TrxnR/NADPH system was able to fully reconstitute the activity of a puri ed poly-S-nitrosated caspase 3 (Fig. 2A,B; Ref. [49]). These observations may be extended to the studies of Zhang et al. who reported that exposure of lung endothelial cells (LEC) to NO results in diminished eNOS activity and decreased expression of Trxn and TrxnR, whereas overexpression of Trxn prevents eNOS inhibition in intact cells [50]. Interestingly, resistance to LPS-induced apoptosis in LEC is manifested by inhibition of caspase 3 but is not apparent until 96 h of endogenous NO generation [51,52]. Hence, it could be speculated that exhaustion of TrxnR activity may be required for the nitrosation of caspase 3 (Scheme 1).

Chelation of metal ions by caspase 3

Recently, Perry et al. have reported that caspase 3 could be inhibited by submicromolar concentrations ofZn2+, which suggests a regulatory role for Zn2+ in modulating the upstream apoptotic machinery; mechanistically, Zn2+ has been proposed to form a complex with caspase 3 [53]. This hypothesis is in agreement with the studies of Tang et al. and Kondo et al. who have demonstrated that the release of Zn2+ from metallothioneins leads to inhibition of caspase 3 in mouse embryonic cells and cultured sheep pulmonary artery endothelial cells, respectively [54,55]. More recently, Sliskovic and Mutus have demonstrated that caspase 3 chelates iron ions with concomitant loss of activity, whereby both EDTA and DTT could reverse this effect [56]. The latter indicates that the chelation of iron ions by caspase 3 was not followed by oxidation of its thiol functions.

Iron complexes containing thiyl and NO ligands

Interactions between iron complexes and NO have been the focus of much research that dates back to the studies of Priestley [57 62]. Recently, Dobry-Duclaux has shown that submicro-molar concentrations of the black Roussin's salt (K[Fe4S3(NO)7]) impede the enzyme alcohol dehydrogenase [63,64], while Gordy and Rexroad demonstrated that complexes of nitric oxide with hemoglobin and cytochrome c exhibit ESR spectra that re ect the electronic structures of the iron atoms in these biologically important molecules [65]. Despite their notorious instability, numerous iron-nitrosyl complexes have been isolated in crystal form and characterized by x-ray analysis and IR, NMR, ESR, and MS spectrometry. Experimental proof has been provided for the formation of iron-nitrosyl complexes in neutral aqueous solutions of Fe2+ containing anionic ligands such as pyrophosphate, adenosine triphosphate (ATP), creatine phosphate, carbonate, maleate, and mercaptans (2-mercaptoethanol, thiourea, dithiols, and cysteine). However, it is noteworthy that most studies have focused on the geometrical and electronic structures of iron-nitrosyl complexes, whereas the (bio)chemical properties of this class of compounds have not been well-characterized.

In recent years, the formation and interactions of iron-nitrosyl complexes in biological matrices have been of particular interest with respect to the transduction of NO signals [16,17,66 69]. In weakly alkaline milieu, cysteine interacts with ferrous ions to form a bis(cysteinato)iron2+ complex [59]. The formation of this complex is paralleled by the appearance of a violet color that gradually fades with the consumption of O2. Simultaneously, cysteine is partially transformed to cystine, but, as long as there remains any thiol acid, the color may be regenerated by O2. This catalytic process has been proposed to occur via three steps: rst, formation of bis(cysteinato)iron2+ complex; second, oxidation of this complex by O2 to a Fe3+ complex, which is the cause of the violet color observed in the catalytic process; and third, an autoreduction of the ferric complex in which the iron is reduced to a divalent state while the thiol sulfur of cysteine is oxidized to thiyl radical cation (LS+ ) with simultaneous breakdown of the complex and fading of the color. In cellular systems, non-heme iron is expected to form complexes with GSH and to exhibit even higher af nity for compounds containing vicinal thiol functions. Bonomi et al. have characterized a series of ferric and ferrous complexes of 6,8-dimercapto-octanoic acid (dihydrolipoic acid; DHLA) and dihydrolipoamide as potential substrates for enzymatic synthesis of iron sulfur clusters, whereas Kijima et al. have reported that these complexes are strong reductants that can convert aromatic NO2 functions to NH2 and A,O-dialkylhydroxylamines to alkylamines and alcohols [70].

Was this article helpful?

0 0

Post a comment