Spectrophotometric and Electron Spin Resonance Studies of NADH2-cytochrome c Reductase Complex

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Abstract
The effect of o-phenanthroline on the activity of NADH2-cytochrome c reductase complex from beef heart muscle mitochondria was investigated. The cytochrome c reductase activity of this complex was o-phenanthroline-sensitive, while the activity of ferricyanide reductase was insensitive to this inhibitor. A marked loss of activity of cytochrome c reductase was observed when the enzyme complex had been incubated with o-phenanthroline for several hours. It does not seem to be caused by the release of non-heme iron. When the enzyme complex was incubated with NADH2 at 0°C, the ESR spectrum, having a signal at g=1.94, due to non-heme iron, distinctly changed with the time of incubation, that is, new ESR signals at g=1.98 and g=1.95 (weak) appeared after 20 min of incubation. These signals disappeared on further incubation for 20 hr and a signal at g=1.93 appeared. The ferricyanide reductase activity decreased to 70% on incubation for 20 min, but after this, the activity did not change during the course of change of the ESR spectrum described above. Ferricyanide seems to react directly with the flavin of the NADH2 dehydrogenase [EC 1.6.99.3], but not through the “ESR-active” non-heme iron. By the spectrophotometric method and ESR technique, it was confirmed that the “ESR-active” non-heme iron and NADH2 dehydrogenase flavoprotein in the enzyme complex are located before the site of inhibition by o-phenanthroline. It was suggested that in the enzyme complex there is an unknown respiratory component having an absorption band at about 465 mμ besides NADH2 dehydrogenase. This unknown component seems to be located after the site of inhibition by o-phenanthroline or Amytal.

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