Experiments were performed to examine the cyanide-binding properties of resting and pulsed cytochrome c oxidase in both their stable and transient turnover states. Inhibition of the oxidation of ferrocytochrome c was monitored as a function of cyanide concentration. Cyanide binding to partially reduced forms produced by mixing cytochrome c oxidase with sodium dithionite was also examined. A model is presented that accounts fully for cyanide inhibition of the enzyme, the essential feature of which is the rapid, tight, binding of cyanide to transient, partially reduced, forms of the enzyme populated during turnover. Computer fitting of the experimentally obtained data to the kinetic predictions given by this model indicate that the cyanide-sensitive form of the enzyme binds the ligand with combination constants in excess of 10(6) M-1 X s-1 and with KD values of 50 nM or less. Kinetic difference spectra indicate that cyanide binds to oxidized cytochrome a33+ and that this occurs rapidly only when cytochrome a and CuA are reduced.

Oxidized cytochrome c oxidase can bind hydrogen peroxide, as evidenced by changes in its spectrum and its ability to use hydrogen peroxide as an electron acceptor in cytochrome c oxidation. The affinity of the oxidized enzyme for hydrogen peroxide is high, with a Kd of less than 10 microM, and the binding is inhibited by ligands of cytochrome a3. Oxidized cytochrome c oxidase, in submitochondrial particles or solubilized in several ionic and nonionic detergents, binds peroxide with comparable affinities. The size of the spectral shift observed upon peroxide binding depends on the pH of the solution and differs in extinction coefficient between preparations, but all preparations tested appeared to bind peroxide. The differences in the magnitude of the spectral shift upon peroxide binding to different preparations suggest that oxidized cytochrome c oxidase as prepared may be made up of more than one species and that the proportion of the species which binds peroxide varies with the preparation. These studies of the binding of peroxide clarify the mechanism by which cytochrome c oxidase catalyzes the reduction of oxygen to water without the formation of free-radical intermediates.

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The suprachiasmatic nucleus (SCN) in the hypothalamus serves as the pacemaker for mammalian circadian rhythms. In a hamster brain slice preparation, the authors were able to record spontaneous activity from SCN cells for up to 4 days in vitro and verify a self-sustained rhythm in firing. The phase of this rhythm was altered by the concentration of glucose in the bathing medium, with time of peak firing advanced for a 20 mM glucose condition and slightly delayed for a 5 mM glucose condition, relative to 10 mM. The advancing effect of 20 mM glucose and the delaying effect of 5 mM glucose were not maintained during a 2nd day in vitro after changing the bathing medium back to 10 mM glucose, thus indicating the effect was not a permanent phase shift of the underlying oscillation. In experiments recording from cell-attached membrane patches on acutely dissociated hamster SCN neurons, exchanging the bathing medium from high (20 mM) to zero glucose increased potassium (K+)-selective channel activity. With inside-out membrane patches, the authors revealed the presence of a glybenclamide-sensitive K+ channel (190 pS) and a larger conductance (260 pS) Ca(2+)-dependent K+ channel that were both reversibly inhibited by ATP at the cytoplasmic surface. Furthermore, 1 mM tetraethylammonium chloride was demonstrated to advance peak firing time in the brain slice in a similar manner to a high concentration of glucose (20 mM). The authors interpret the result to imply that SCNs are sensitive to glucose, most probably via ATP modulation of K+ channel activity in these neurons. Tonic modulation of K+ channel activity appears to alter output of the pacemaker but does not reset the phase.

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