pH-induced alteration and oxidative destruction of heme in purified chromaffin granule cytochrome b(561): implications for the oxidative stress in catecholaminergic neurons

Abstract

The transmembrane hemoprotein, cytochrome b(561) (b(561)), in the neuroendocrine secretory vesicles is shown to shuttle electrons from the cytosolic ascorbate (Asc) to the intravesicular matrix to provide reducing equivalents for the dopamine beta-monooxygenase (DbetaM) reaction. Intravesicular Asc may also play a role in relieving catecholamine-induced oxidative stress in catecholaminergic neurons. In the present study, we have examined the alteration of purified oxidized b(561) (b(561,ox)) under mild alkaline conditions to probe the structural and functional characteristics of the protein, using UV-vis and EPR spectroscopic and kinetic techniques. Our results show that low spin heme in oxidized b(561) (b(561,ox)) readily transforms to an altered high spin form and then slowly to an Asc nonreducible form, in a pH-, temperature-, and time-dependent manner, which can be described by single-exponential rate equations, A(t) = A(o)(1- e (-kt)) and A(t) = A(o)e(-kt), respectively. More than half of the Asc nonreducible altered b(561) could be converted back to the native b(561) by pH adjustment followed by dithionite reduction, suggesting the reversibility of the process. The heme center of the transformed Asc nonreducible protein is completely bleached instantaneously by dithionite in the presence of atmospheric oxygen, which appears to be mediated by molecular oxygen and/or hydrogen peroxide. These results demonstrate that the heme centers of the protein are susceptible to the pH-induced alteration and oxidative destruction, raising some questions regarding the proposed one alkaline labile, two-heme model of b(561) [Tsubaki, M.; Nakayama, M.; Okuyama, E.; Ichikawa, Y. (1997) J. Biol. Chem. 272, 23206-23210]. The pH-induced alteration and the destruction of heme under oxidative conditions may play a significant role in the amplification of oxidative stress in catecholaminergic neurons.