Mitchell Fry

Cardiolipin requirement by cytochrome oxidase and the catalytic role of phospholipid

Abstract An effective method is described for the almost complete removal of phospholipid from cytochrome oxidase, with a consequent parallel decrease in enzymic activity. Replenishment of lipid-depleted cytochrome oxidase with purified phospholipids indicates an absolute requirement of enzymic activity for cardiolipin. This catalytic role of cardiolipin is distinguishable from the more general effects of phospholipids and detergents on cytochrome oxidase activity.

Energized transport of cations by cytochrome oxidase

Summary Cytochrome oxidase, when incorporated into a phospholipid liposome, can mediate the energy-dependent transport of monovalent and divalent cations and amino acids. Cation uptake is accompanied by anion uptake. Little specificity for the transportable cation seems to exist, and under appropriate conditions all cations tested may be equally transported.

Intrinsic coupling in cytochrome oxidase nature and stoichiometry of the coupling reactions

Summary The aerobic oxidation of ferrocytochrome c has been studied in lysolecithin dispersions of cytochrome oxidase and in Ca 2+ -state mitochondria by oxygen pulsing of the reaction systems monitored with electrodes appropriate for measuring the concentrations of O 2 , K + and H + . When oxygen is introduced, there is a surge of K + into the particle followed by an efflux of K + from the particle as the oxygen tension diminishes. At the end of the pulse the original level of K + in the medium is restored. Concomitant with K + influx, protons are released into the medium and, concomitant with K + efflux, protons are taken up from the medium. The inverse relation between K + efflux and proton influx is an expression of cation/proton exchange. The values for the K + /e and H + /e ratios have been determined by two independent methods and shown to be close to 1. The U-shaped profile for the changes in K + and H + concentrations during an oxygen pulse experiment provides clear evidence that electron flow in cytochrome oxidase can be coupled to cyclical transport of monovalent cations such as K + . The so-called uncoupled respiration of cytochrome oxidase can now be identified as coupled cyclical transport of cations.

Studies on the resolution of cytochrome oxidase

Cytochrome oxidase has been resolved in acetic acid and high salt/detergent media. In 0.5% acetic acid, the smaller subunits of the enzyme are selectively extracted with retention of an insoluble protein fraction containing subunits I–IV, VII. This fraction retains all the heme and copper of the original enzyme in a spectrally unaltered state, and possesses enzymic activity comparable to the unresolved enzyme. The further removal of subunit IV from this fraction results in migration of heme and copper and modification of their spectral characteristics. Resolution of the enzyme in a high salt/detergent medium extracts smaller subunits (V–VII) together with subunit IV and some heme and copper. The heme associated with this enzymically active extract has spectral characteristics that are partially suggestive of hemea3. It is suggested that the fraction of subunits I–IV, VII, resolved in dilute acetic acid, may represent the limit of resolution of the cytochrome oxidase complex that remains actively and spectrally indistinguishable from the original enzyme.

Energized cation transport by complex III (ubiquinone-cytochrome C reductase).

Abstract Energized cyclical and net transport of cations and anions has been demonstrated in liposomes containing Complex III using a protamine-aggregation technique. Energization with reduced ubiquinone induces a rapid ion uptake followed by a more gradual efflux upon exhaustion of the available reductant. Both monovalent and divalent cations are transported, but at relatively high concentrations divalent cations are transported in preference to monovalent cations. Results are consistent with a cation:electron ratio of unity.

Resolution and reconstitution of cytochrome oxidase

Summary Cytochrome oxidase has been resolved into two fractions by extraction with formic acid. An insoluble-fraction (subunits I–III) retains the heme and phospholipid of the original enzyme and a soluble-fraction (subunits IV–VII) retains the copper. Recombination of the two enzymatically inactive fractions in the presence of sodium deoxycholate results in the expression of full enzymic activity.

The polypeptide composition of subunit I of beef heart cytochrome oxidase.

Abstract It has been shown that subunit I of cytochrome oxidase (∼ MWt. 40,000) can be resolved into a number of smaller polypeptides. This resolution apparently occurs through two stages with the generation of polypeptides of approximate molecular weights of 20,000 and 8,500.

On The Mechanism of Energy Coupling in Cytochrome Oxidase

Highly purified cytochrome oxidase from beef heart mitochondria without any supplementation catalyzes the coupling of the oxidation of ferrocytochrome c by O2 to the transport of monovalent or divalent cations (Green et al., 1980; Fry and Green, 1980a). This finding established that cytochrome oxidase was a complete energy-coupling system and that coupling took only one form—the coupling of electron transfer to cation transport. Cytochrome oxidase was ideally suited to probe the mechanism of energy coupling. The components and structure of the electron transfer chain were well defined, the subunit structure of the complex was firmly established, the stoichiometry of the oxidative reaction (oxidation of ferrocytochrome c by O2) was known, the assay of cytochrome oxidase activity was sufficiently selective that it was possible to measure this activity as readily in mitochondria as in dispersions of the purified oxidase, and finally and most importantly ion transport is the least complicated expression of a coupled process.