The Mechanism For Assembly Of Dnic With Thiolcontaining Ligands

The two pathways described in the previous section yield DNIC complexes with {Fe(NO)2}7 configuration. The mechanism for the assembly of this complex is not just simple liganding of nitrosyl and thiol ligands to the iron, but must necessarily involve some redox transformation of the constituents. The experimental evidence for redox activity is clear: The synthesis proceeds in the absence of oxygen and starts with ferrous iron in d6 state (non-heme ferric iron is not capable of NO binding). Nitrosyl liganding should lead to a diamagnetic {Fe(NO)2}8 configuration, but EPR experiments show the formation of the paramagnetic {Fe(NO)2}7 species instead. Second, quantitative studies [77] show that three NO molecules are needed for the formation of a single DNIC. Third, the synthesis proceeds under the release of one molecule of N2O per two DNIC complexes [85]. This N2O represents nitrogen in a lower oxidation state than the original NO and is usually taken as a manifestation of the formation of an intermediate HNO or NO- species. The formation of N2O and consumption of NO remind us of the well-known dismutation reaction of NO in the gas phase under high pressure. This asymmetrical reaction has products in lower as well as higher oxidation state than the original NO (cf Chapter 1)

In dilute solutions, this dismutation reaction is kinetically forbidden and cannot play a significant role. However, the above evidence suggests that ferrous iron initiates a disproportionation reaction of the form 2NO ^ NO+ + NO- [84] in the liquid phase, where the nitroxyl anion NO- leads to subsequent formation of N2O. Such disproportionation of NO is known to be mediated by a variety of metal complexes with iron, cobalt, copper or ruthenium [86]. The analogous disproportionation reaction leaves the dinitrosyl complex in {Fe(NO)2}7 state:

2L- + 2NO + Fe2+ ^ 2L-{Fe(NO)2}8 (assembly of diamagnetic complex) 2L-{Fe(NO)2}8 + H++ NO ^ 2L-{Fe(NO)2}7 + HNO (disproportionation) 2HNO ^ N2O + H2O (the formation of nitrous oxide)

The precise mechanism for the disproportionation step is not known, but a possible mechanism might proceed as below (Scheme 3) [46,71]:

Fe2^le- -» XFe2+ — >e2+ + HNO^ x Fe+ + /(N2O+H2O) L-/ N NO L-/ VNO- L-/ L/ ^ NO+ \+ NO Net process: Fe2+ + 3NO + 2L- + H+ -» {(L-)2Fe+ (NO+)2} + /(N2O+H2O)

Scheme 3.

We propose that the d-orbitals of the iron mediate this disproportionation by coupling the unpaired electrons of the nitrosyls. The appearance of HNO in the above equations reflects the fact that this protonated nitroxyl rather than NO- is the principal species at physiological pH (early reports of pKa ~ 4.7 have been significantly revised upward to ca 11 [87]). NO- is isoelectronic with dioxygen and can exist as a spin triplet ground state or spin singlet excited state. The triplet ground state has a low rate of protonation due to restrictions imposed by spin conservation [62b,87] (for properties of the nitroxyl anion cf Chapter 1).

According to Scheme 3, the formation of DNIC from ferrous iron and NO proceeds through three steps [46]. First is the formation of paramagnetic MNIC with {FeNO}7 configuration:

The next step involves the inclusion of a second NO ligand, the reduction of this ligand to nitroxyl and the release of this nitroxyl moiety from the complex after protonization as shown:

(L-)2Fe+—NO+ + NO ^ (L-)2Fe2+—(NO+, NO-) + H+ ^ (L-)2Fe2+—NO+ + HNO

The mononitrosyl complex at this stage is diamagnetic with {FeNO}6 configuration. The final step is binding the third NO molecule to give DNIC with {Fe(NO)2 }7, which is paramagnetic:

(L-)2Fe2+—NO+ + NO ^ (L-)2Fe2+—(NO+, NO) ^ (L-)2Fe+—(NO+)2

It should be noted that diamagnetic mononitrosyl complex can also be formed by the inclusion of a nitrosyl ligand in a ferrous dithiocarbamate complex [46].

A similar situation may be expected if the DNIC is assembled from nitrosothiols RS—NO instead of free NO molecules, if the redox disproportionation reaction between two RS—NO

molecules is proposed [46,71]:

One-electron reduction/oxidation of RS—NO molecules leads to the appearance of instable products of the reaction. In the solution without iron, the process can be negligible. However, the disproportionation of RS—NO molecules can be sharply accelerated being catalyzed by ferrous iron. The experiments demonstrate that addition of iron to solutions of Cys-NO or GS-NO resulted in rapid formation of paramagnetic DNIC: The rate constant is comparable to that of the DNIC formation from iron and nitric oxide [46]. So, a possible mechanism might proceed as below [46,71]:

Scheme 4.

The assembly of the intermediate diamagnetic complex is reversible as it does not involve any electronic rearrangement which prevents release of the RS—NO moieties. The disproportionation with asymmetrical release of RS- and RS^ is irreversible, however, and drives the reaction to full synthesis of DNIC in {Fe(NO)2}7 state. Subsequently, the thiyl radicals RS^ combine into a disulfide.

Scheme 4 predicts that two Cys-NO molecules are needed for the formation of one iron-dinitrosyl complex, in accordance with quantitative estimates from experimental data [54]. The experiments were performed on Cys-DNIC as synthesized by NO treatment of cys-teine and ferrous iron solution in 10 mM HEPES buffer, pH 7.4. Upon rapid acidification of the DNIC solution to pH 1.5 with HCl, the solution turned from green to pink color and lost the EPR absorption from DNIC. The optical absorption confirmed the formation of Cys-NO, the quantity being equal to that of the initial Cys-DNIC. Subsequent rapid raising the pH back to initial value (pH 7.4) with NaOH restored the green color at reduced intensity. Optical absorption confirmed the reconstruction of around 50% of the initial Cys-DNIC. Repeating this cycle of acidification subsequently induced loss of ca 50% of the Cys-DNIC, with corresponding reductions in the amounts of Cys-NO. It means that two Cys-NO molecules were consumed for the reconstruction of one DNIC complex induced by increasing the pH from acid to neutral values.

It should be remarked that the above reactions were studied in vitro under strict exclusion of oxygen. Such conditions are never strictly met in actual biological systems. Therefore, it should be kept in mind that additional pathways might exist for in vivo formation of DNIC. In the presence of oxygen, diamagnetic DNIC with {Fe(NO)2}8 will be oxidized rapidly to paramagnetic {Fe(NO)2}7 (see previous sections).


As mentioned earlier, low-molecular-weight DNICs with thiol-containing ligands have much higher stability against oxygen from ambient air than DNICs with non-thiolic ligands. This difference reflects the difference in binding strength of the nitrosyl ligands to the iron. This binding is enhanced significantly by the coordination of thiol ligands to the iron [39]. Low stability of DNIC with non-thiol containing ligands could be due to the hydrolysis of the nitrosonium moieties by hydroxyl anions (Scheme 5) [46,88]:

Scheme 5.

The interaction could lead to irreversible accumulation of nitrite ions. With respect to DNIC with thiol-containing ligands, due to high affinity of thiols to nitrosonium cations NO+ [89] the interaction between them could lead to the formation of S-nitrosothiols (Scheme 6) by the release of ferrous iron:


Scheme 6.

The partial decomposition of DNIC proceeds until a quasi-equilibrium between DNIC and its constituents is established. This reaction equilibrium between DNIC, NO, ferrous irons, RS- and RS—NO is considered in detail in Chapter 11 of this book.

However, the assembly of DNIC from ferrous iron + RS—NO leads to the oxidation of thiols to thiyl radicals and formation of disulfides even at anaerobic conditions (Scheme 3). As a result, the concentration of thiols decreases with time to levels where DNIC starts to dimerize as diamagnetic DNIC (see above). Subsequent reinforcement of the thiol concentration reverts to monomeric state. These transformations are illustrated in Fig. 25 showing the formation of Cys-DNIC in an anaerobic solution of Cys-NO + ferrous iron with low cysteine:iron ratio. The sharp fall of the g = 2.03 signal with time reflects the loss of paramagnetic DNIC and can be reversed by the addition of excess cysteine [46]. This recovery of the 2.03 signal is even achieved after solutions of the dimeric DNIC were stored anaerobically for several hours and attest to the high stability of the complexes.

Upon exposure to oxygen, the loss of paramagnetism is significantly accelerated for weakly binding and thiol ligands. For example, the exposure of 1 mM of paramagnetic phosphate-DNIC in 100 mM phosphate buffer (pH = 7.4) to ambient air leads to full disappearance of the EPR signal from the solution, characteristic of the complex, within 10 min [86]. Similar behavior was characteristic of other DNIC with non-thiol-containing ligands (citrate, ascorbate, water and other; Vanin, unpublished data). With thiolate ligands like cysteine, the transformation of monomeric Cys-DNIC to dimeric Cys-DNIC is accelerated by oxygen, evidently due to oxidation of thiol compounds with oxygen. For example, exposure of 0.2 mM monomeric Cys-DNIC to ambient air leads to complete loss of paramagnetism within 40 min. The iron has been sequestered as diamagnetic dimeric MNIC, because the EPR intensity may be fully recovered by the addition of 10 mM of L-cysteine [55]. Longer exposure to oxygen subsequently leads to irreversible loss of DNIC: After storage of dimeric DNIC open to ambient air for 4 h, only 50% of the original intensity may be recovered by the addition of L-cysteine. Still, the long lifetime attests to a high stability of dimeric DNIC in particular against oxidation (Vanin, unpublished data).

Oxidation of the nitrosyl ligands is another possible pathway. French investigators have studied DNIC with bidentate NN ligands such as 2,2'-bipiridine, 4,4-dimethyl-2,2'-bipiridine, 1-10-phenanthroline, with bidentate PN ligands such as its 2-(diphenylphosphino) and 2-(diphenylphosphine oxide) derivatives, and with bidentate phosphorus ligands PP such as diphosphines 1,2-bis(diphenylphosphine)-ethane or trans- or cis- 1,2-bis(diphenylphosphine)ethylene [90-93]. Exposure to dioxygen transforms the DNIC into the nitrato iron complexes. The structures of these nitrato complexes were determined as Fe(NO3)2Cl(NN), Fe(NO3)2Cl(OPN) or [FeCl4][Fe(NO3)2Cl(OPPPO)2], respectively. Thus, it shows oxidation of the nitrosyl ligands and formation of nitrato ligands in the iron complexes. The mechanism of the process is under study now. It was noted that these complexes have sufficient electron density on the iron to transfer oxygen and allow oxidation of various organic compounds including diphenylphospine ligands [91].

Depending on the final redox state of the iron, some nitrato iron complexes would be paramagnetic, others EPR-silent state. This may explain the experimental fact that superoxide readily reacts with DNIC-cysteine to an EPR-silent product [94]. The same observation applies to DNIC bound to serum albumin. Moreover, the antioxidant features are characteristic of DNICs [94-98]. The transformation of NO to nitrato ligands by superoxide was proposed [91] to explain the observed loss of paramagnetism. The reaction would initially produce peroxynitrite ligated to the iron, followed by isomerization to nitrate. Significantly, thiosulfate was observed [99] to protect DNIC against degradation by superoxide. This protection applied to Cys-DNIC as well as DNIC bound to serum albumin, and was attributed to transfer of the Fe(NO)2 moiety to the thiosulfate ligand, thereby converting the DNIC to a more stable form. Such protection is highly significant for possible therapeutical applications of thiosulfate-DNIC as vasodilator or inhibitor of platelet aggregation, where the presence of superoxide radicals is known to play an important role.

This section discussed only few of the possible reaction mechanisms of the oxidative agents with DNIC complexes. Future investigations will provide deeper insight into the complex chemistry and functions of DNIC in biosystems.

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