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 \u27autoreduction\u27 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 α and β 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 a3. Under certain conditions when both CO and oxygen are present, a peroxide adduct derived from oxygen reduction can be observed. This \u27607 nm complex\u27, described in 1981 by Nicholls and Chanady, 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, autoreduction 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 a3 to cytochrome a and cytochrome c

The Mechanism by which Oxygen and Cytochrome \u3ci\u3ec\u3c/i\u3e Increase the Rate of Electron Transfer from Cytochrome a to Cytochrome a3 of Cytochrome \u3ci\u3ec\u3c/i\u3e 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/Cu(A) to cytochrome a3/Cu(B)), but assumes a relatively rapid two-electron transfer between cytochrome a/Cu(A) and cytochrome a3/Cu(B) and a thermodynamic equilibrium in the \u27resting\u27 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 \u3ci\u3ec\u3c/i\u3e 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 hammer-head 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

Carbon Monoxide Promotes Respiratory Hemoproteins Iron Reduction Using Peroxides as Electron Donors

The physiological role of the respiratory hemoproteins (RH), hemoglobin and myoglobin, is to deliver O2 via its binding to their ferrous (FeII) heme-iron. Under variety of pathological conditions RH proteins leak to blood plasma and oxidized to ferric (FeIII, met) forms becoming the source of oxidative vascular damage. However, recent studies have indicated that both metRH and peroxides induce Heme Oxygenase (HO) enzyme producing carbon monoxide (CO). The gas has an extremely high affinity for the ferrous heme-iron and is known to reduce ferric hemoproteins in the presence of suitable electron donors. We hypothesized that under in vivo plasma conditions, peroxides at low concentration can assist the reduction of metRH in presence of CO. The effect of CO on interaction of metRH with hydrophilic or hydrophobic peroxides was analyzed by following Soret and visible light absorption changes in reaction mixtures. It was found that under anaerobic conditions and low concentrations of RH and peroxides mimicking plasma conditions, peroxides served as electron donors and RH were reduced to their ferrous carboxy forms. The reaction rates were dependent on CO as well as peroxide concentrations. These results demonstrate that oxidative activity of acellular ferric RH and peroxides may be amended by CO turning on the reducing potential of peroxides and facilitating the formation of redox-inactive carboxyRH. Our data suggest the possible role of HO/CO in protection of vascular system from oxidative damage

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 μM) 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\u27s 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\u27s effects are tissue-specific. Analysis of the effects of heme\u27s degradation products on SCN PER2::LUC rhythms indicated that they probably were not responsible for heme\u27s 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