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

The transmembrane hemoprotein, cytochrome b561 (b561), 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 β-monooxygenase (DβM) 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 b561 (b561,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 b561 (b561,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, At = Ao(1 − e-kt) and At = Aoe-kt, respectively. More than half of the Asc nonreducible altered b561 could b...