Reductive nitrosylation of ferric carboxymethylated-cytochrome c

Horse heart carboxymethylated-cytc (CM-cytc) displays myoglobin-like properties due to the cleavage of the heme-Fe-Met80 axial bond. Here, reductive nitrosylation of CM-cytc(III) between pH 8.5 and 9.5, at T = 20.0 degrees C, is reported. Under anaerobic conditions, the addition of NO to CM-cytc( III) leads to the transient formation of CM-cytc(III)-NO in equilibrium with CM-cytc(II)-NO+. In turn, CM-cytc(II)-NO+ is converted to CM-cytc(II) by OH- -based catalysis. Then, CM-cytc(II) binds NO very rapidly leading to CM-cytc(II)-NO. Kinetics of NO binding to CM-cytc(III) is independent of the ligand concentration, k values ranging between 3.6 +/- 0.4 s(-1) and 7.1 +/- 0.7 s(-1). 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-cytc(III)-NO to CM-cytc(II)-NO is rate-limited by the OH- -mediated reduction of CM-cytc(II)-NO+ (h(OH-)= (1.2 +/- 0.1) x 10(3) M-1. s(-1)). Lastly, the very fast nitrosylation of CM-cytc(II) takes place, values of l(on) ranging between 5.3 x 10(6) M-1. s(-1) and 1.4 x 10(7) M-1. s(-1). These results indicate that CM-cytc behaves as the cardiolipin-cytc complex highlighting the role of the sixth axial ligand of the heme-Fe atom in the modulation of the metal-based reactivity.