Nitric oxide induces Zn2+ release from metallothionein by destroying zinc‒sulphur clusters without concomitant formation of S-nitrosothiol

Abstract

The reaction of nitric oxide (NO) with metallothionein (MT) has been investigated at neutral pH under strictly anaerobic conditions. It is observed that NO mediates zinc release from MT by destroying zinc-sulphur clusters, but that it does not by itself S-nitrosylate MT in contrast to common belief. Zinc release and loss of thiolate groups under anaerobic conditions is found to be much slower than under aerobic conditions. The observed percentage loss of Zn(2+) and thiolate groups after 3 h of NO treatment are 62 and 39%, respectively. The reaction of NO with cysteine is reinvestigated and it is found that cysteine is quantitatively converted to cystine after 5 min of NO treatment at pH 7.3. At lower pH, a much lower rate of conversion is observed confirming the base-catalysed nature of the reaction of NO with thiols. On the basis of these results, a reaction mechanism involving electrophilic attack of NO on thiolate groups and subsequent formation of a nitrogen-centred radical, MTSN(. )OH, as intermediate is proposed for the reaction of NO with MT that leads to zinc release.

Nitric Oxide and Zinc Homeostasis in Acute Lung Injury

“…Subsequently, Spahl and coworkers (89) noted that iNOS-derived NO increased nuclear Zn and that this increase appeared to require the translocation of MT from cytoplasm to nucleus. We (90,91) and others (92,93) confirmed these observations and demonstrated that: (1 ) S-nitrosation caused conformational changes of MT (via fluorescence resonance energy transfer techniques; Figure 1) in intact pulmonary endothelium consistent with zinc release (4, 94); (2 ) NO caused an increase in free zinc in pulmonary artery endothelial cells (21,95); and (3 ) MT was the requisite target for NO resulting in such changes in free zinc (5). The metal status of MT was critical for resultant NO-mediated changes in that: (1 ) NO did not cause release of zinc in a cell in which most of the MT was in its apo form (5); and (2 ) Cu-MT was also nitrosated and depending upon the copper status and the amount of NO exposure, CuMT served as a copper chaperone for apo-ZnSOD (96) or a source of Fenton reactive copper (91).…”

“…The metal status of MT was critical for resultant NO-mediated changes in that: (1 ) NO did not cause release of zinc in a cell in which most of the MT was in its apo form (5); and (2 ) Cu-MT was also nitrosated and depending upon the copper status and the amount of NO exposure, CuMT served as a copper chaperone for apo-ZnSOD (96) or a source of Fenton reactive copper (91). S-nitrosation: (1 ) requires the presence of molecular oxygen (92,97), (2 ) is modified by redox status of the environ- Figure 1. A schema of a fluorescence resonance energy transfer (FRET) capable reporter molecule (MT) and full spectral report of a single sheep pulmonary artery endothelial cell exposed to the NO donor, L-S-nitrosocysteine ethyl ester (L-SNCEE).…”

Modulation of nitric oxide‐mediated metal release from metallothionein by the redox state of glutathione in vitro

“…Due to the different metal affinities for zinc and cadmium in the two separate domains [13], the b-domain has been implicated in zinc homeostasis and the tight binding of cadmium in the a-domain was proposed to be responsible for the role of MTs in heavy metal detoxification. In addition, it has been reported that MTs act as radical scavengers under oxidative stress [20][21][22].Another possible key player in the role of MTs in signal transduction might be nitric oxide (NO), which was shown recently, both in vitro [23][24][25] and in vivo [26][27][28][29], to interact with MTs and thereby releases bound zinc and cadmium. The importance of MTs in NO-induced changes in intracellular zinc homeostasis has been reported by St Croix et al [30].…”

“…Another possible key player in the role of MTs in signal transduction might be nitric oxide (NO), which was shown recently, both in vitro [23][24][25] and in vivo [26][27][28][29], to interact with MTs and thereby releases bound zinc and cadmium. The importance of MTs in NO-induced changes in intracellular zinc homeostasis has been reported by St Croix et al [30].…”

“…This activity relies on the suppression of the expression and activity of inducible nitric oxide synthase (iNOS) by zinc [32,33], released from MT under the influence of nitric oxide (NO), and the scavenging of NO through covalent binding to MTs [23] to form S-nitrosothiols. Such a role of MTs in the inflammatory response has been corroborated by the significantly altered inflammatory behavior during experimental autoimmune encephalomyelitis [34] observed in MT deficient mice.…”