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Fig. 11. Formation of Cys-DNIC as detected with EPR in HEPES (100 mM, pH 7.4) at room temperature. (A) Kinetics of Cys-DNIC formation in the mixture of 1 mM cysteine, 400 ^M Cys-NO, 50 ^M Fe2+ and 250 ^M sodium citrate. (B) The EPR spectrum of the solution at the end of reaction at 1500 s and (C) EPR spectrum of sample (B) after addition of 20 mM cysteine. (From Ref. [19].)

2 {(RS-)2Fe+(NO+)2}+ « " {(RS-)2Fe+(NO+)2}22+ +RS-

Scheme 6. The equilibrium between monomeric and dimeric forms of thiol-DNIC is determined by the quantity of excess free thiol.

of magnitude lower: EPR showed that only a small fraction of GS-DNIC was of paramagnetic monomeric form, whereas the majority was dimeric diamagnetic DNIC. This finding is in line with Chapter 2, where the monomeric/dimeric ratio at neutral pH was found to depend on the Fe2+/thiol ratio, and on the thiol used. For our conditions, cysteine preferably forms monomeric DNIC, whereas other thiols like glutathione preferably form dimeric DNIC.

The experimental data could be explained by the following mechanism for the formation of Cys-DNIC from Cys-NO and iron (Scheme 7):

First rapid step is the formation of intermediate paramagnetic Cys-MNIC: (RS-)„Fe2+ + NO-RS^ ^^ (RS-)„Fe2+ NO-RS^

Second step is the formation of DNIC by transfer of a nitrosonium from the nitrosothiol to the iron:

—> (RS-)2Fe+(NO+)2 + RS-Taken together, the net reaction can be presented as:

Scheme 7. The formation of DNIC with cysteine from Cys-NO and ferrous iron.

Taken into account the quasi-equilibrium between DNIC and its constituents (Scheme 5), the net process can be presented as follows:

Fe2+

In this form, the result resembles the reductive degradation of RS-NO catalyzed by copper ions (Scheme 1). However, copper acts by true redox cycling, whereas iron acts by cycling through a DNIC intermediate.

It is significant that the upgrade from MNIC to DNIC is achieved by transfer of a nitrosonium moiety from an S-nitrosothiol rather than by a free NO. Therefore, the formation of DNIC does not require successive capture of two free NO molecules, which would be difficult to achieve at the nanomolar NO concentrations under normal physiological conditions. Instead, the second nitrosyl moiety is taken as a nitrosonium from an S-nitrosothiol, which is found at far higher concentrations in the low micromolar range (cf Chapters 9 and 10).

For cysteine ligands, all steps in the process of Cys-DNIC formation could be studied in vitro. We propose that the same reaction equilibria also apply to mixtures of iron with other thiols and their S-nitrosothiols.

A less stable DNIC with phosphate appeared when the solutions of Fe2+-citrate in 100 mM phosphate buffer (pH 7.4) were mixed with Cys-NO (Fig. 12A). The EPR signal of the complex recorded at ambient temperature had a singlet shape at g = 2.032 with septet hyperfine structure from nitrogen atoms of two NO ligands and phosphorus atoms of two phospate ligands (Fig. 12D). The rapid formation of DNIC with phosphate was followed by its rapid decay.

Similar kinetics was observed if NO-proline was added instead of Cys-NO (Fig. 12C). Low stability of DNIC with phosphate could be due to hydrolysis of NO+ groups in the complex as shown in Scheme 8:

Thiol-DNIC has far higher stability and longer lifetime in aqueous solution. We attribute this to electrostatic binding of thiol anions with NO+ moieties and formation of coordinated RS-NOs. However, due to electron migration from Fe+ to one NO+, one RS-NO ligand can be decomposed resulting in the chemical equilibrium (Scheme 9) similar to that shown in Scheme 5:

The mechanism of RS-NO decomposition catalyzed by iron and presented in Scheme 7 can be true for the proteins containing thiol ligands. Evidently due to high affinity of protein-bound

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