Reductive nitrosylation of ferric carboxymethylated-cytochrome c

Abstract

Horse heart carboxymethylated-cyt[Formula: see text] (CM-cyt[Formula: see text] displays myoglobin-like properties due to the cleavage of the heme-Fe-Met80 axial bond. Here, reductive nitrosylation of CM-cyt[Formula: see text](III) between pH 8.5 and 9.5, at [Formula: see text] 20.0 C, is reported. Under anaerobic conditions, the addition of NO to CM-cyt[Formula: see text](III) leads to the transient formation of CM-cyt[Formula: see text](III)-NO in equilibrium with CM-cyt[Formula: see text](II)-NO[Formula: see text]. In turn, CM-cyt[Formula: see text](II)-NO[Formula: see text] is converted to CM-cyt[Formula: see text](II) by OH[Formula: see text]-based catalysis. Then, CM-cyt[Formula: see text](II) binds NO very rapidly leading to CM-cyt[Formula: see text](II)-NO. Kinetics of NO binding to CM-cyt[Formula: see text](III) is independent of the ligand concentration, [Formula: see text] values ranging between 3.6 ± 0.4 s[Formula: see text] and 7.1 ± 0.7 s[Formula: see text]. This indicates that the formation of the CM-cytc(III)-NO complex is rate-limited by the cleavage of the weak heme-Fe(III) distal bond (likely Lys79). The conversion of CM-cyt[Formula: see text](III)-NO to CM-cyt[Formula: see text](II)-NO is rate-limited by the OH[Formula: see text]-mediated reduction of CM-cyt[Formula: see text](II)-NO[Formula: see text] ([Formula: see text] (1.2 ± 0.1) × 103 M[Formula: see text].s[Formula: see text]. Lastly, the very fast nitrosylation of CM-cyt[Formula: see text](II) takes place, values of [Formula: see text] ranging between[Formula: see text]5.3 × 106 M[Formula: see text].s[Formula: see text] and 1.4 × 107 M[Formula: see text].s[Formula: see text]. These results indicate that CM-cyt[Formula: see text] behaves as the cardiolipin-cyt[Formula: see text] complex highlighting the role of the sixth axial ligand of the heme-Fe atom in the modulation of the metal-based reactivity.