Fig. 1. Kinetics of the decomposition of 2 mM Cys-NO at ambient temperature as monitored by the optical absorption at 548 nm. (Curve 1) Without any additives, (Curve 2) With 2 mM cysteine and (Curve 3) With 2 mM neocuproine. The arrows indicate time points at which 0.5 mM Fe2+ was added. (From Ref. [25].)

to the 1.8 mM solution of DNIC with cysteine containing 7.2 mM cysteine at pH = 7.4 led to practically full transformation of Cys-DNIC into neocuproine-DNIC [25].

Interestingly, neocuproine is capable of protecting Cys-NO against the catalytic decomposition by ferrous iron (Fig. 1) [25]: The experiments were performed on 20 mM solutions of Cys-NO in 10 mM HEPES buffer, pH 7.4. The addition of 2 mM cysteine to the solution prevented rapid (for 10 min) decomposition of Cys-NO (Fig. 1, curves 1,2).

The decomposition was initiated at this condition by bolus addition of 0.5 mM ferrous iron and could be followed by optical absorption. The decomposition was quite fast and completed after ca 5 min (Fig. 1, curve 2). The decomposition could be inhibited if 2 mM neocuproine was added prior to ferrous iron (Fig. 1, curve 3). In presence of Cys-NO and neocuproine, the addition of ferrous iron induces formation of significant quantities of DNIC. Aliquots frozen immediately after the addition of iron showed intense EPR absorption by neocuproine-DNIC, observable at g = 2.02 (Fig. 2, curve b). The nature of this complex was unambiguously identified by the broadening of the EPR spectrum when ferrous 57Fe iron was added to the solution. This broadening is characteristic for the hyperfine structure (HFS) from this iron isotope (Fig. 2, curve a).

These EPR experiments have revealed two important properties of neocuproine: First and foremost, that neocuproine is capable of chelating ferrous Fe2+ as well as Cu+. Second, that the presence of free NO or nitrosothiols results in the formation of neocuproine-DNIC. The paramagnetism of the neocuproine-DNIC shows that the complex has an electronic configuration where the unpaired electrons of the nitrosyl ligands are largely transferred towards the iron. In Enemark-Feltham notation, the electronic state of the complex is written

Fig. 2. The shape of the EPR spectra from 57Fe-(or 56Fe)-DNIC neocuproine (A,B). Doublet spectra above spectra A is low-field part of the latter recorded at higher amplification. The spectra were recorded at 77 K. (From Ref. [25].)

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