The effect of formate on cytochrome [formula omitted] and on electron transport in the intact respiratory chain

Abstract

1. Formate inhibits cytochrome c oxidase activity both in intact mitochondria and submitochondrial particles, and in isolated cytochrome aa 3 . The inhibition increases with decreasing pH, indicating that HCOOH may be the inhibitory species. 2. Formate induces a blue shift in the absorption spectrum of oxidized cytochrome aa 3 (a 3+a 3 3+ ) and in the half-reduced species ( a 2+a 3 3+ ). Comparison with cyanide-induced spectral shifts, towards the red, indicates that formate and cyanide have opposite effects on the aa 3 spectrum, both in the fully oxidized and the half-reduced states. The formate spectra provide a new method of obtaining the difference spectrum of a 3 2+ minus a 3 3+ , free of the difficulties with cyanide (which induces marked high → low spin spectral shifts in cytochrome a 3 3+ ) and azide (which induces peak shifts of cytochrome a 2+ towards the blue in both α- and Soret regions). 3. The rate of formate dissociation from cytochrome a 2+a 3 3+- HCOOH is faster than its rate of dissociation from a 3+a 3 3+- HCOOH , especially in the presence of cytochrome c. The K i for formate inhibition of respiration is a function of the reduction state of the system, varying from 30 mM (100% reduction) to 1 mM (100% oxidation) at pH 7.4, 30 °C. 4. Succinate-cytochrome c reductase activity is also inhibited by formate, in a reaction competitive with succinate and dependent on [formate] 2. 5. Formate inhibition of ascorbate plus N,N,N′,N′- tetramethyl-p- phenyl-enediamine oxidation by intact rat liver mitochondria is partially released by uncoupler addition. Formate is permeable through the inner mitochondrial membrane and no differences in ‘on’ or ‘off’ inhibition rates were observed when intact mitochondria were compared with submitochondrial particles. 6. NADH-cytochrome c reductase activity is unaffected by formate in submitochondrial particles, but mitochondrial oxidation of glutamate plus malate is subject both to terminal inhibition at the cytochrome aa 3 level and to a slow extra inhibition by formate following uncoupler addition, indicating a third site of formate action in the intact mitochondrion.