Fig. 8. EPR spectra at room temperature of aliquots of 500 ^M Fe2+, 10 mM cysteine, and 400 ^M Cys-NO in HEPES (100 mM, pH 7.4). Panel A: 10 min after mixing. Panel B: 20 min after mixing. The triplet signal at g = 2.04 is ferrous Cys-MNIC. The singlet at g = 2.03 is Cys-DNIC. (From Ref. [19].)

intermediate step. Once Cys-DNIC had been formed, its lifetime was significantly shortened to a few minutes by the presence of deoxy-Hb (Fig. 10C,D). We attribute the decay of Cys-DNIC to scavenging of NO ligands from the DNIC by deoxy-Hb. This interpretation is compatible with the concept of a kinetic equilibrium between DNIC and its constituents as given in Scheme 5. This equilibrium implies the presence of a small quantity of free NO molecules in the solution:

The kinetics of Fig. 10D show that the destruction of Cys-DNIC by deoxy-Hb does not start immediately, but start after an "induction period" of about 3 min. We attribute this "induction period" to the presence of excess Cys-NO. The latter provides a pool of NO to regenerate Cys-DNIC, thereby maintaining Cys-DNIC at a stationary level until the Cys-NO is exhausted.

It is remarkable that the presence of a NO scavenger like deoxy-Hb does not influence the formation of DNIC from Cys-NO and ferrous iron. In particular, the yields of Cys-DNIC yields are unaffected (Fig. 10D). It proves that the mechanism of formation does not require free NO radicals, and does not involve the equilibrium reaction of Scheme 5. The reaction

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