ATP synthesis driven by α-keto acid-stimulated alternative oxidase in pea leaf mitochondria

Abstract

The electrical potential difference (ΔΨ) generation and ATP synthesis due to α-keto acid-stimulated alternative oxidase activity in pea leaf mitochondria, energized by malate plus glutamate, was studied. In the absence of α-keto acids, ΔΨ was ca. 60% collapsed by KCN or antimycin A (AA), while the remaining part (ca. 30%) was dissipated by salicyl-hydroxamic acid (SHAM). The presence of α-keto acids (pyruvate or iodoacetate), known to stimulate the alternative oxidase, caused a decrease in the KCN- or AA-sensitive component of ΔΨ (ca. 30%), which was accompanied by an increase in the SHAM-sensitive one. When mitochondria were energized by exogenous NADH, succinate or duroquinol, ΔΨ was completely collapsed by KCN or AA, either in the presence or absence of α-keto acids. ADP partially collapsed the SHAM-sensitive component of ΔΨ, and oligomycin restored this dissipation. In the presence of pyruvate or iodoacetate, ATP content in KCN-treated mitochondria was ca. 40% of control, while that in SHAM plus KCN-inhibited mitochondria was negligible. ADP stimulated oxygen uptake only in the presence of KCN (respiratory control ratio=1.7), but not in the presence of KCN plus SHAM. It is concluded that α-keto acids, stimulating the oxidation of NAD-linked substrates through the alternative oxidase, lead to an increase in the SHAM-sensitive component of ΔΨ via complex I, thus increasing the contribution of this pathway to ATP synthesis when the cytochrome oxidase is restricted.