Complex III Releases Superoxide to Both Sides of the Inner Mitochondrial Membrane

Abstract

Mechanisms of mitochondrial superoxide formation remain poorly understood despite considerable medical interest in oxidative stress. Superoxide is produced from both Complexes I and III of the electron transport chain, and once in its anionic form it is too strongly charged to readily cross the inner mitochondrial membrane. Thus, superoxide production exhibits a distinct membrane sidedness or "topology." In the present work, using measurements of hydrogen peroxide (Amplex red) as well as superoxide (modified Cypridina luciferin analog and aconitase), we demonstrate that Complex I-dependent superoxide is exclusively released into the matrix and that no detectable levels escape from intact mitochondria. This finding fits well with the proposed site of electron leak at Complex I, namely the iron-sulfur clusters of the (matrix-protruding) hydrophilic arm. Our data on Complex III show direct extramitochondrial release of superoxide, but measurements of hydrogen peroxide production revealed that this could only account for ϳ50% of the total electron leak even in mitochondria lacking CuZn-superoxide dismutase. We posit that the remaining ϳ50% of the electron leak must be due to superoxide released to the matrix. Measurements of (mitochondrial matrix) aconitase inhibition, performed in the presence of exogenous superoxide dismutase and catalase, confirmed this hypothesis. Our data indicate that Complex III can release superoxide to both sides of the inner mitochondrial membrane. The locus of superoxide production in Complex III, the ubiquinol oxidation site, is situated immediately next to the intermembrane space. This explains extramitochondrial release of superoxide but raises the question of how superoxide could reach the matrix. We discuss two models explaining this result.

An active mitochondrial biogenesis occurs during dendritic cell differentiation

“…It has in fact been reported that Complex I generated superoxide is released exclusively into the matrix, together with 50% of the superoxide produced by Complex III (Muller et al, 2004). On the other hand, the key role of the mitochondrial SOD is illustrated by the lethal phenotype of mice lacking the matrix superoxide dismutase (Sod2) gene (Li et al, 1995).…”

Redox homeostasis and age‐related deficits in neuromuscular integrity and function

“…Resting and contracting skeletal muscle produces superoxide via different pathways, and schematic Figures 3 and 4 depict the various sites and mechanisms that have been proposed for RONS generation in skeletal muscle. Briefly, superoxide is generated by the mitochondrial electron transport chain including complex I, complex III76, 77 and, recently, complex II78, 79, 80; the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzymes including NOX2, NOX4, DUOX1 and DUOX255, 56, 59, 81; xanthine oxidase82, 83; and the lipoxygenases (LOXs),84 which are linked to arachidonic acid released by the phospholipase A 2 enzymes85, 86 (for a detailed review, see Ref. [46].…”

Mitochondrial complex III regulates hypoxic activation of HIF

“…41,43,[47][48][49] Unlike complexes I and II, which generate superoxide into the mitochondrial matrix, [52][53][54][55] complex III can also release superoxide into the mitochondrial intermembrane space and subsequently, into the cytosol. [56][57][58] The Q cycle is initiated when complex I and complex II transfer electrons to reduce the lipid moiety ubiquinone to ubiquinol (Figure 3). Ubiquinol then enters the Q cycle (Figure 3).…”