The main findings of the present study are as the following: 1) mitochondrial DLDH could be reversibly inactivated by Angeli’s salt; 2) the inactivation was a targeting rather than a random process as peroxynitrite, at similar concentrations, did not show any inhibitory effect on DLDH activity; 3) the RNS that actually inactivated DLDH was HNO derived from Angeli’s salt; and 4) the inactivation likely involved formation of s-nitrosothiols on DLDH.
It is noteworthy that when incubated with whole mitochondria, Angeli’s salt seemed to target DLDH. In contrast, peroxynitrite lacked this targeting ability in the presence of other mitochondrial proteins (); although peroxynitrite, when incubated with purified DLDH, could indeed inactivate the enzyme (unpublished data, this laboratory). Such observations suggest that peroxynitrite is consumed by other mitochondrial proteins before it can get to DLDH when whole mitochondria are treated by peroxynitrite. Alternatively, peroxynitrite, at the same concentration as that of Angeli’s salt, may not be as effective as Angeli’s salt in inactivating DLDH. Therefore, no inhibitory effect of peroxynitrite could be detected under our experimental conditions. Regardless, it is suffice to say that inactivation of DLDH by Angeli’s salt is a targeting process.
Angeli’s salt is a unique donor of RNS in that it can decompose readily at physiological pH with the production of two reactive nitrogen species, i.e., HNO and nitrite. Nevertheless, most of Angeli’s salt actions on proteins have been attributed to HNO 
. In the present study, we also found that it was indeed HNO, but not nitrite, that actually inactivated DLDH (). This result is therefore in consistent with those by others that HNO is the actual species that imposes the biochemical and pharmacological effects of Angeli’s salt [36, 37]
. It should be noted that nitric oxide can also be produced when Angeli’s salt is incubated with ferricyanide 
. Nonetheless, nitric oxide generated this way apparently had no detectable inhibitory effect on DLDH activity ().
While we determined that DLDH inactivation by Angeli’s salt likely involved the formation of s-nitrosothiols, we were unable to determine which of DLDH’s redox-sensitive cysteine residues that were actually nitrosylated and led to decrease in DLDH activity. Our attempt to analyze modified cysteine residues by mass spectrometry techniques failed mainly because the peptide containing the two cysteine residues could not be recovered following HPLC and mass spectrometry analysis. Therefore, the redox cysteine residue(s) that was nitrosylated could only be speculated. DLDH has two cysteine residues at its active center that are engaged in catalysis, namely, cys45 and cys50 
. During catalysis, cys45 is the substrate-binding residue while cys50 is the FAD interacting residue. Studies by others have indicated that cys45 is more reactive than cys50 toward thiol-reactive reagents 
. In glutathione reductase whose structure is similar to that of DLDH, the substrate binding cysteine residue could be nitrosylated and sulfenated upon treatment with nitric oxide donors 
. Such studies suggest that the nitrosylated cysteine residue on DLDH is likely that of cys45 following treatment with Angeli’s salt.
It is well-established that protein s-nitrosylation is a reversible process and is involved not only in oxidative damage 
, but also in regulation and expansion of protein function 
. For example, s-nitrosylation can contribute to protein misfolding and neuronal synaptic damage 
; s-nitrosylation of mitochondrial proteins can also be involved in ischemic preconditioning that protects hearts against subsequent, severe ischemic injury 
. While the physiological and pathophysiological significance of DLDH nitrosylation remains to be determined, such modification will certainly impair the enzyme’s function that may further perturb mitochondrial oxidative phosphorylation. It should be noted that while it has been previously reported that DLDH underwent s-nitrosylation [58, 59]
, none of these earlier studies examined the relationship between s-nitrosylation and loss of enzyme activity. In contrast, our present data suggest that nitrosylation was likely responsible, at least in part, for the loss of DLDH activity.
It should be pointed out that the ascorbate/biotin switch method may yield a false-positive signal in detection of protein S-nitrosylation 
. Therefore, it might be argued that our observation of DLDH nitrosylation may have nothing to do with the observed reversible inactivation of DLDH function. This is the reason why we have only cautiously concluded that S-nitrosylation is probably the cause of DLDH reversible inactivation. A definitive conclusion can only be reached when both control and treated samples undergo the procedures of ascorbate reduction and biotin switch, whereby no positive or less intensive nitrosylation signals will be detected in the control samples. On the other hand, it is also possible that formation of protein sulfenic acids (S-sulfenation) could be partially responsible for the reversible inactivation of DLDH function observed in the present study as protein sulfenic acids can be readily derived from protein S-nitrosothiols 
. If this is indeed the case, DLDH nitrosylation induced by Angeli’s salt would be only a transient form of DLDH modification and may only be considered partially responsible for the loss in DLDH activity. In other words, both S-nitrosylation and S-sulfenation may be involved in loss of DLDH activity induced by Angeli’s salt. Regardless, whether Angeli’s salt can induce DLDH sulfenic acid formation under our experimental conditions will need to be further investigated.
In summary, our study has indicated that brain mitochondrial DLDH could be inactivated in a dose-dependent manner by Angeli’s salt and the inactivation was caused by HNO. Our results have also revealed that DLDH inactivation by HNO was a targeting process and was accompanied by HNO-induced S-nitrosylation. Overall, the present study provides insights into the mechanisms of DLDH inactivation by Angeli’s salt, albeit that further studies are needed to determine which cysteine residues are nitrosylated.