Evidence for the occurrence in submitochondrial particles of a dual respiratory chain containing different forms of cytochrome b

Abstract

1. Addition of ascorbate + N, N, N′, N′-tetramethyl- p-phenylenediamine (TMPD) to beef-heart submitochondrial particles (Mg-ATP particles) in the presence of KCN causes extensive reduction of cytochromes c 1 + c and a + a 3, and a partial reduction of cytochrome b. Addition of ATP results in an increased cytochrome b reduction, with a slight decrease in the level of reduced cytochrome a + a 3. Subsequent addition of NAD + causes a further substantial reduction of cytochrome b and a partial oxidation of cytochrome a + a 3. The total amount of cytochrome b reduced at this stage is close to that obtained by adding NADH or succinate to the particles in the presence of KCN. 2. Cytochrome b reduced upon the addition of TMPD and NAD + has an absorption maximum at 562 nm, and that reduced upon the addition of ATP at 565 nm, with a shoulder at 558 nm. All three phases of cytochrome b reduction are inhibited by antimycin, and the ATP- and NAD +-induced phases are also inhibited by FCCP and oligomycin. The NAD +-induced cytochrome b 562 reduction is ATP-dependent, and is abolished by rotenone and by pyruvate + lactate dehydrogenase, indicating that it proceeds by way of NADH generated through reverse electron transport via cytrochrome b 565 (+ b 558). The effect of NAD + in inducing cytochrome b 562 reduction is duplicated by fumarate in a non-additive fashion. 3. The three phases of cytochrome b reduction are accompanied by roughly proportional extents of reduction of ubiquinone and flavorprotein. The amounts of cytochrome b, ubiquinone and flavoprotein, reduced upon the addition of ascorbate + TMPD, ATP and NAD +, are close to the total enzymically (NADH and succinate) reducible contents of these components in the particles. 4. The results are interpreted to indicate that submitochondrial particles contain a dual respiratory chain, one including cytochrome b 565 (+ b 558) and a functional Coupling Site II, and another, including cytochrome b 562 and no functional Coupling Site II. The possible significance of these results for the functional organization of mitochondria is discussed.