Suprachiasmatic nucleus neurons are glucose sensitive.

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.

Inhibitors of the mitochondrial cytochrome b-c1 complex inhibit the cyanide-insensitive respiration of Trypanosoma brucei.

The cyanide-insensitive respiration of bloodstream trypomastigote forms of Trypanosoma brucei (75 +/- 8 nmol O2 min-1(mg protein)-1) is completely inhibited by the mitochondrial ubiquinone-like inhibitors 2-hydroxy-3-undecyl-1,4-naphthoquinone (UHNQ) and 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT). The Ki values for UHDBT (30 nM) and UHNQ (2 microM) are much lower than the reported Ki for salicylhydroxamic acid (SHAM) (5 microM), a widely used inhibitor of the cyanide-insensitive oxidase. UHNQ also stimulated the glycerol-3-phosphate-dependent reduction of phenazine methosulfate, demonstrating that the site of UHNQ inhibition is on the terminal oxidase of the cyanide-insensitive respiration of T. brucei. These results suggest that a ubiquinone-like compound may act as an electron carrier between the two enzymatic components of the cyanide-insensitive glycerol-3-phosphate oxidase.

Functional equivalence of monomeric (shark) and dimeric (bovine) cytochrome c oxidase.

Cytochrome c oxidase isolated from hammerhead shark red muscle is monomeric in relation to the dimeric form of isolated bovine cytochrome c oxidase but in other ways bears a close resemblance to the enzyme isolated from mammalian tissue [1, 2]. Comparative studies of shark and bovine cytochrome c oxidase were extended to address the degree of functional similarity between the monomeric (shark) and dimeric (bovine) enzymes in the kinetics of peroxide binding and in the extent to which the catalytic action of the enzymes in vesicles can establish a proton gradient. Although the kinetics of peroxide binding and the proton pumping processes are complex, the dimeric and monomeric forms are quite similar with respect to these functional attributes. The kinetic heterogeneity of the process of peroxide binding is expressed in the shark enzyme as well as in the bovine enzyme, and both types of enzymes in vesicles can generate transmembrane proton gradients. On this basis we conclude that the dimeric state of isolated cytochrome c oxidase from mammalian sources is not essential for its function in vitro.

Heme reversibly damps PERIOD2 rhythms in mouse suprachiasmatic nucleus explants.

The hypothalamic suprachiasmatic nucleus (SCN), which in mammals serves as the master circadian pacemaker by synchronizing autonomous clocks in peripheral tissues, is composed of coupled single-cell oscillators that are driven by interlocking positive/negative transcriptional/translational feedback loops. Several studies have suggested that heme, a common prosthetic group that is synthesized and degraded in a circadian manner in the SCN, may modulate the function of several feedback loop components, including the REV-ERB nuclear receptors and PERIOD2 (PER2). We found that ferric heme (hemin, 3-100 microM) dose-dependently and reversibly damped luminescence rhythms in SCN explants from mice expressing a PER2::LUCIFERASE (PER2::LUC) fusion protein. Inhibitors of heme oxygenases (HOs, which degrade heme to biliverdin, carbon monoxide, and iron) mimicked heme's effects on PER2 rhythms. In contrast, heme and HO inhibition did not damp luminescence rhythms in thymus and esophagus explants and had only a small effect on PER2::LUC damping in spleen explants, suggesting that heme's effects are tissue-specific. Analysis of the effects of heme's degradation products on SCN PER2::LUC rhythms indicated that they probably were not responsible for heme's effects on rhythms. The heme synthesis inhibitor N-methylprotoporphyrinIX (NMP) lengthened the circadian period of SCN PER2::LUC rhythms by about an hour. These data are consistent with an important role for heme in the circadian system.

Phosphate binding to Escherichia coli alkaline phosphatase. Evidence for site homogeneity.

The reversible, noncovalent binding of inorganic phosphate to Escherichia coli alkaline phosphatase at pH 8 has been examined by equilibrium dialysis at two temperatures and two ionic strengths. Binding occurs with a stoichiometry of two phosphate ions per dimeric enzyme molecule and a single dissociation constant that is not very sensitive to temperature or ionic strength. These results contradict published evidence for anti-cooperative binding of inorganic phosphate to alkaline phosphatase. Reasons are presented for believing that the apparent anti-cooperativity reported by other workers is artifactual.

Cytochrome oxidase as a proton pump.

The general structure of cytochrome oxidase is reviewed and evidence that the enzyme acts as a redox-linked proton pump outlined. The overall H+/e- stoichiometry of the pump is discussed and results [Wikström (1989), Nature 338, 293] which suggest that only the final two electrons which reduce the peroxide adduct to water are coupled to protein translocated are considered in terms of the restrictions they place on pump mechanisms. "Direct" and "indirect" mechanisms for proton translocation are discussed in the context of evidence for redox-linked conformational changes in the enzyme, the role of subunit III, and the nature of the CuA site.

Cytochrome c oxidase binding of hydrogen peroxide.

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.

A high-affinity protein stain for western blots, tissue prints, and electrophoretic gels.

A method for protein staining using copper phthalocyanine 3,4',4'',4'''-tetrasulfonic acid tetrasodium salt is described. The procedure is applicable to protein blots and tissue prints, as well as to polyacrylamide and agarose gels. It is also simple, involving only application of the stain and rinsing. For protein blots and tissue prints the staining is rapid, taking less than 1 min to completion, and more sensitive than any previously described dye-based nonspecific protein staining system. The staining is easily reversible, requiring only a change in pH to remove the dye.

Carbon monoxide-driven reduction of ferric heme and heme proteins.

Oxidized cytochrome c oxidase in a carbon monoxide atmosphere slowly becomes reduced as shown by changes in its visible spectra and its reactivity toward oxygen. The "auto-reduction" of cytochrome c oxidase by this procedure has been used to prepare mixed valence hybrids. We have found that this process is a general phenomenon for oxygen-binding heme proteins, and even for isolated hemin in basic aqueous solution. This reductive reaction may have physiological significance. It also explains why oxygen-binding heme proteins become oxidized much more slowly and appear to be more stable when they are kept under a CO atmosphere. Oxidized alpha and beta chains of human hemoglobin become reduced under CO much more slowly than does cytochrome c oxidase, where the CO-binding heme is coupled with another electron accepting metal center. By observing the reaction in both the forward and reverse direction, we have concluded that the heme is reduced by an equivalent of the water-gas shift reaction (CO + H2O----CO2 + 2e- + 2H+). The reaction does not require molecular oxygen. However, when the CO-driven reduction of cytochrome c oxidase occurs in the presence of oxygen, there is a competition between CO and oxygen for the reduced heme and copper of cytochrome alpha 3. Under certain conditions when both CO and oxygen are present, a peroxide adduct derived from oxygen reduction can be observed. This "607 nm complex," described in 1981 by Nicholls and Chanady (Nicholls, P., and Chanady, G. (1981) Biochim. Biophys. Acta 634, 256-265), forms and decays with kinetics in accord with the rate constants for CO dissociation, oxygen association and reduction, and dissociation of the peroxide adduct. In the absence of oxygen, if a mixture of cytochrome c and cytochrome c oxidase is incubated under a CO atmosphere, auto-reduction of the cytochrome c as well as of the cytochrome c oxidase occurs. By our proposed mechanism this involves a redistribution of electrons from cytochrome alpha 3 to cytochrome alpha and cytochrome c.

The mechanism by which oxygen and cytochrome c increase the rate of electron transfer from cytochrome a to cytochrome a3 of cytochrome c oxidase.

When cytochrome c oxidase is isolated from mitochondria, the purified enzyme requires both cytochrome c and O2 to achieve its maximum rate of internal electron transfer from cytochrome a to cytochrome a3. When reductants other than cytochrome c are used, the rate of internal electron transfer is very slow. In this paper we offer an explanation for the slow reduction of cytochrome a3 when reductants other than cytochrome c are used and for the apparent allosteric effects of cytochrome c and O2. Our model is based on the conventional understanding of cytochrome oxidase mechanism (i.e. electron transfer from cytochrome a/CuA to cytochrome a3/CuB), but assumes a relatively rapid two-electron transfer between cytochrome a/CuA and cytochrome a3/CuB and a thermodynamic equilibrium in the "resting" enzyme (the enzyme as isolated) which favors reduced cytochrome a and oxidized cytochrome a3. Using the kinetic constants that are known for this reaction, we find that the activating effects of O2 and cytochrome c on the rate of electron transfer from cytochrome a to cytochrome a3 conform to the predictions of the model and so provide no evidence of any allosteric effects or control of cytochrome c oxidase by O2 or cytochrome c.

Kinetics of reduction of cytochrome c oxidase by dithionite and the effect of hydrogen peroxide.

The reduction of cytochrome c oxidase by dithionite was reinvestigated with a flow-flash technique and with varied enzyme preparations. Since cytochrome a3 may be defined as the heme in oxidase which can form a photolabile CO adduct in the reduced state, it is possible to follow the time course of cytochrome a3 reduction by monitoring the onset of photosensitivity. The onset of photosensitivity and the overall rate of heme reduction were compared for Yonetani and Hartzell-Beinert preparations of cytochrome c oxidase and for the enzyme isolated from blue marlin and hammerhead shark. For all of these preparations the faster phase of heme reduction, which is dithionite concentration-dependent, is almost completed when the fraction of photosensitive material is still small. We conclude that cytochrome a3 in the resting enzyme is consistently reduced by an intramolecular electron transfer mechanism. To determine if this is true also for the pulsed enzyme, we examined the time course of dithionite reduction of the peroxide complex of the pulsed enzyme. It has been previously shown that pulsed cytochrome c oxidase can interact with H2O2 and form a stable room temperature peroxide adduct (Bickar, D., Bonaventura, J., and Bonaventura, C. (1982) Biochemistry 21, 2661-2666). Rather complex kinetics of heme reduction are observed when dithionite is added to enzyme preparations that contain H2O2. The time courses observed provide unequivocal evidence that H2O2 can, under these conditions, be used by cytochrome c oxidase as an electron acceptor. Experiments carried out in the presence of CO show that a direct dithionite reduction of cytochrome a3 in the peroxide complex of the pulsed enzyme does not occur.

A re-examination of the reactions of cyanide with cytochrome c oxidase.

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.

Paradoxical effects of methylmercury on the kinetics of cytochrome c oxidase.

A stoichiometric amount of methylmercuric chloride substantially inhibits cytochrome c oxidase function under steady-state turnover conditions, where the enzyme is using its substrates, cytochrome c and oxygen, rapidly and continuously. Under these conditions, a reduction in activity of approximately 40% is observed. This is in accord with the results of Mann and Auer [Mann, A.J., & Auer, H.E. (1980) J. Biol. Chem. 255, 454-458], who used mercuric chloride and ethylmercuric chloride. Paradoxically, we found that addition of methylmercuric chloride can increase the activity of cytochrome c oxidase during its initial substrate utilization. This rate enhancement, measured under conditions where the enzyme cycles only a few times, is maximal for the resting state of the enzyme. "Pulsed" cytochrome c oxidase (i.e., enzyme that has been recently reduced and reoxidized) is considerably activated with respect to the resting enzyme, showing faster turnover rates (Antonini, 1977; Brunori et al., 1979). No significant rate enhancement upon treatment with methylmercuric chloride is seen in initial substrate utilization if the enzyme is pulsed immediately before the assay. The apparently contradictory effects of methylmercuric chloride on the resting and pulsed states of the oxidase under low turnover conditions may be reconciled by a model in which mercurial binding greatly stabilizes the enzyme in a state resembling that of the pulsed enzyme. A decrease in conformational flexibility may be the basis of the mercurial-induced diminution in activity of the enzyme during steady-state turnover conditions.