Hydroxylamine-induced oxidation of ferrous CO-bound carboxymethylated-cytochrome c

Abstract

The hexa-coordinated metal center of horse heart cyt[Formula: see text] (cyt[Formula: see text] is at the root of its low reactivity. In contrast, carboxymethylated cyt[Formula: see text] (CM-cyt[Formula: see text] displays myoglobin-like properties. Herein, kinetics of CO binding to ferrous CM-cyt[Formula: see text] (CM-cyt[Formula: see text](II)) and of the irreversible oxidation of ferrous carbonylated CM-cyt[Formula: see text] (CM-cyt[Formula: see text](II)-CO) by hydroxylamine (HA), at pH 5.8 and 20.0 [Formula: see text]C, are reported. HA irreversibly oxidizes CM-cyt[Formula: see text](II)-CO with the 1:2 stoichiometry leading to the formation of the ferric species (CM-cyt[Formula: see text](III)) without the observation of intermediates. Present data indicate that: (i) the rate of CO dissociation from CM-cyt[Formula: see text](II)-CO represents the rate-limiting step of HA-mediated oxidation of the carbonylated metal center, (ii) the fast oxidation of CM-cyt[Formula: see text](II)-CO from HA reflects the penta-coordination of the transient CM-cyt[Formula: see text](II) species, (iii) the HA-catalyzed conversion of CM-cyt[Formula: see text](II)-CO to CM-cyt[Formula: see text](III) could proceed via the geminate mechanism, (iv) values of the second-order rate constants for the carbonylation and the HA-mediated oxidation of ferrous heme-proteins are linearly correlated reflecting the penta- or hexa-coordination of the metal center, the free energy for the in-plane positioning of the heme-Fe atom in the unliganded species, and the arrangement of the distal portion of the heme pocket that affects ligand and/or electron transfer.