Oxidation State Of Cytochrome Research Articles

Kinetics of Binding Processes of Cytochrome C onto Liposome Surfaces

Association processes of cytochrome C (from horse heart) onto surfaces of liposomes which were composed of L-α-dimyristoylphosphatidylglycerol and L-α-dimyristoylphosphatidylcholine were investigated by the stopped-flow technique. The association processes were divided into two relaxation processes: the faster process whose apparent rate constant monotonously increased with the concentration of cytochrome C, and the slower process whose rate constant showed a saturation behavior. When the number of binding sites on the liposome surface was taken into account ( N = 1870), the corrected association rate constant of the faster process ( k corr. = 1.7 × 10 9 M -1s -1) was 2.8% of the theoretical value ( k theor. = 6.0 × 10 10 M -1 s -1) for a binary collision, probably due to a disadvantageous surface-searching and dehydration processes on the liposome and protein surfaces. The effects of deformability of liposomes, components in the lipid bilayers, and oxidation state of cytochrome C on the rate constants for faster and slower steps were examined. The temperature effects on the rate constants were also discussed.

Mutation of Tyrosine-67 to Phenylalanine in Cytochrome c Significantly Alters the Local Heme Environment

The high resolution three-dimensional atomic structures of the reduced and oxidized states of the Y67F variant of yeast iso-1-cytochrome c have been completed. The conformational differences observed are localized directly in the mutation site and in the region about the pyrrole A propionate. Shifts in atomic positions are largely restricted to nearby amino acid side-chains, whereas little perturbation of the polypeptide chain backbone is observed. One prominent difference between the variant and wild-type structures involves a substantial increase in the size of an already existing internal cavity adjacent to residue 67. This same cavity contains an internally bound water molecule (Wat166), which is found in all eukaryotic cytochromes c for which structures are available. In the reduced Y67F mutant protein a second water molecule (Wat300) is observed to reside in this enlarged internal cavity, assuming a position approximately equivalent to that of the hydroxyl group of Tyr67 in the wild-type protein. A further consequence of this mutation is the alteration of the hydrogen bond network between Tyr67, Wat166 and other nearby residues. This appears to be responsible for the absence of oxidation state dependent changes in polypeptide chain flexibility observed in the wild-type protein. Furthermore, loss of the normally resident Tyr67 OH to Met80 SD hydrogen bond leads to a significantly lower midpoint reduction potential. These results reaffirm proposals that both Tyr67 and Wat166 play a central role in stabilizing the alternative oxidation states of cytochrome c.

Recovery of energy metabolism in rat brain after carbon monoxide hypoxia.

Carbon monoxide (CO) may inhibit mitochondrial electron transport in the brain and increase the toxic effects of the gas. This hypothesis was investigated in anesthetized rats during CO exposure and recovery at either normobaric or hyperbaric O2 concentrations. During exposure and recovery, we measured the oxidation level of cerebrocortical cytochrome c oxidase by differential spectroscopy and biochemical metabolites known to reflect aerobic energy provision in the brain. CO exposure (HbCO = 71 +/- 1%) significantly decreased blood pressure and cytochrome oxidation level. Cerebral ATP was maintained while lactate/pyruvate, glucose, and succinate rose, and phosphocreatine (PCr) fell, relative to control (P less than 0.05). Intracellular pH (pHi) calculated from the PCr equilibrium also declined during the exposures. During recovery, HbCO fell more rapidly at hyperbaric than at normobaric O2 levels, but returned to 10% or less in both groups by 45 min. Cytochrome oxidation state improved to 80% of control after 90 min at normobaric O2, but recovered completely after hyperbaric O2 (P less than 0.05). In normobaric O2, PCr and pHi continued to fall for 45 min after CO exposure and did not recover completely by 90 min. PCr and pHi in animals after hyperbaric O2 improved within 45 min, but also remained below control at 90 min. These data indicate that intracellular uptake of CO can impair cerebral energy metabolism, despite the elimination of HbCO from the blood.

Resolution of the electronic transitions of cytochrome c oxidase: evidence for two conformational states of ferrous cytochrome alpha.

Second-derivative absorption spectra are reported for a variety of oxidation and ligation states of bovine cytochrome c oxidase (ferrocytochrome-c:oxygen oxidoreductase, EC 1.9.3.1). The high resolving power of the second-derivative method allows us to assign the individual electronic transitions of cytochrome alpha and cytochrome alpha 3 in many of these states. In the fully reduced enzyme, one observes a single electronic transition at 444 nm, corresponding to the Soret transition for both ferrous cytochrome alpha and ferrous cytochrome alpha 3. When the cytochrome alpha 3 site is occupied by an exogenous ligand (CN or CO), one observes two absorption bands assignable to the ferrous cytochrome alpha chromophore, one at ca, 443 nm and the other at ca, 450 nm. The appearance of the 450-nm band is dependent only on ligand occupancy at the cytochrome alpha 3 site and not on the oxidation state of the cytochrome alpha 3 iron. These results can be interpreted either in terms of a heterogeneous mixture of two ferrous cytochrome alpha conformers in the cytochrome alpha 3-ligated enzyme or in terms of a reduction in the effective molecular symmetry of the ferrous cytochrome alpha site that results in a lifting of the degeneracy of the lowest unoccupied molecular orbital associated with the Soret pi,pi* transition of cytochrome alpha. In either case, the present data indicate that ferrous cytochrome alpha can adopt two distinct conformations. One possible structural difference between these two states could be related to differences in the strength of hydrogen bonding between the ferrous cytochrome alpha formyl oxygen and a proton donor from an unidentified amino acid side chain of the enzyme. The implications of such modulation of hydrogen-bond strength are discussed in terms of possible mechanisms of proton translocation and electron transfer in the enzyme.

Dynamic mechanisms of cardiac oxygenation during brief ischemia and reperfusion

Myocardial oxygenation may be altered markedly by changes in tissue blood flow. During brief ischemia and reperfusion produced by transient occlusion of the left anterior descending artery in 10 open-chest dogs, changes in the oxygenation of tissue hemoglobin (Hb) plus myoglobin (Mb) and the oxidation-reduction (redox) state of mitochondrial cytochrome aa3 were monitored continuously using near-infrared spectroscopy. The nondestructive optical technique indicated that coronary occlusion produced an abrupt drop in tissue oxygen stores (tHb02 + Mb02), tissue blood volume (tBV), and the oxidation level of cytochrome aa3. Changes in the cytochrome oxidation state were related inversely to transmural collateral blood flow within the ischemic region (r = 0.77) measured with radiolabeled microspheres. Furthermore, there was a direct relationship (r = 0.91) between collateral blood flow and the tissue level of desaturated Hb and Mb (tHb + Mb). Reperfusion after 2 min of ischemia led to a synchronous overshoot of baseline in coronary flow and tBV followed by supranormal increases in tHb + Mb02 and the oxidation level of cytochrome aa3. The tHb + Mb level increased transiently during reperfusion. This response correlated inversely with collateral flow during ischemia (r = 0.91). Accordingly, the time required to reach peak tHb + Mb levels was shortest in dogs with high collateral flows (r = 0.75). Thus collateral blood flow partially sustains myocardial oxygenation during coronary artery occlusion and influences tissue reoxygenation early during reperfusion.

Cerebrocortical oxygenation and ventilatory response during sustained hypoxia

Cerebrocortical oxygenation was monitored in 8 healthy adults during exposure to sustained isocapnic hypoxia. Subjects were maintained at an arterial oxygen saturation (Sa O 2 ) of 80% for 12 min with a rebreathing circuit while cerebrocortical oxygenation was assessed non-invasively using near-infrared (NIR) spectroscopy to measure changes in the oxidation state of cytochrome a, a 3 (Cyt a, a 3) and changes in cortical blood volume (tBV). During sustained hypoxia, subjects demonstrated a biphasic ventilatory response. The mean ventilation (V̇ E) peak response was 255% of baseline at an average of 3.4±0.5 min (mean±SE) after the initiation of hypoxia. A subsequent significant attenuation of V̇ E to 163% ( P<0.05) of baseline occurred after an additional 8.6 min. NIR monitoring revealed a significant ( P<0.05) decrease in oxidized Cyt a, a 3 as well as a significant ( P<0.05) increase in tissue blood volume (tBV) at the time of peak V̇ E. Both Cyt a, a 3 and tBV remained stable during the remainder of the hypoxic period, despite attenuation of V̇ E during sustained hypoxia. The data suggest that cerebrocortical oxygenation and blood flow remain constant when the ventilatory attenuation is observed during sustained hypoxia.

Primary intermediate in the reaction of oxygen with fully reduced cytochrome c oxidase.

The primary intermediate in the reaction of oxygen with cytochrome c oxidase was generated by photodissociating carbon monoxide in a continuous flow rapid mixing apparatus. The presence of the primary intermediate was confirmed by a comparison of the iron-dioxygen stretching frequency with that obtained in the reaction of oxygen with the mixed-valence enzyme. For both of these preparations, the Fe-O2 stretching mode is detected at 568 cm-1, the same frequency as that found in oxyhemoglobin and oxymyoglobin. These data illustrate that the primary intermediate may be generated and detected at room temperature in the fully reduced enzyme and that the oxidation state of cytochrome a does not affect the structure of the iron-dioxygen complex. By following the changes in the intensity of the Fe-O2 stretching mode in the resonance Raman spectrum as a function of time, the first-order rate constant for the decay of the primary intermediate was found to be 3.5 x 10(4) s-1 (t1/2 = 20 microseconds).

Primary intermediate in the reaction of mixed-valence cytochrome c oxidase with oxygen

The reaction of dioxygen with mixed-valence cytochrome c oxidase was followed in a rapid-mixing continuous-flow apparatus. The optical absorption difference spectrum and a kinetic analysis confirm the presence of the primary oxygen intermediate in the 0-100-microseconds time window. The resonance Raman spectrum of the iron-dioxygen stretching mode (568 cm-1) supplies evidence that the degree of electron transfer from the iron atom to the dioxygen is similar to that in oxy complexes of other heme proteins. Thus, the Fe-O2 bond does not display any unique structural features that could account for the rapid reduction of dioxygen to water. Furthermore, the frequency of the iron-dioxygen stretching mode is the same as that of the primary intermediate in the fully reduced enzyme, indicating that the oxidation state of cytochrome a plays no role in controlling the initial properties of the oxygen binding site.

Metal site cooperativity within cytochrome oxidase.

Low temperature (9-15 K) EPR of isolated bovine heart cytochrome oxidase titrated potentiometrically in the presence of azide reveals the formation of two distinct species of low-spin cytochrome a3(III)-azide which differ in redox properties and g values. Both species are formed with characteristic midpoint potentials during the course of oxidative titration and disappear at higher potentials. The signal appearing at lower potential has principal g values 2.88, 2.19, and 1.64; that appearing at higher potential has g values 2.77, 2.18, and 1.74. A good fit to the experimental data (per cent of cytochrome present in a given paramagnetic state versus oxidation potential) was obtained with a model whereby the gz = 2.88 species arises from cytochrome a3(III)-azide with cytochrome a reduced, which is converted to the gz = 2.77 species upon oxidation of cytochrome a. Potentiometric titration of cytochrome oxidase in the presence of cyanide produces two low-spin heme EPR signals attributable to cytochrome a3(III)-cyanide which are incompletely resolved, but are distinguishable nonetheless. The low-potential signal has peak amplitude at gz = 3.63 and a long high-field tail; this resonance has been seen by other workers in the partially reduced enzyme (DerVartanian, D. V., Lee, I. Y., Slater, E. C., and van Gelder, B. F. (1974) Biochim. Biophys. Acta 347, 321-327). The high-potential signal is much more symmetric about its peak amplitude, which is at approximately 10 G higher field with gz = 3.61. As with the azide complex, the titration behavior in the presence of 2 mM KCN is adequately simulated by assuming that the appearance of the two species is a function of the oxidation state of cytochrome a. Like the a3-azide signals, the a3-cyanide signals disappear upon further oxidation with some characteristic midpoint potential. If the disappearance of the a3-ligand signals with increasing potential is assumed to be the result of antiferromagnetic (or ferromagnetic) coupling of a3(III) (S = 1/2) to CuB(II) (S = 1/2), then cooperativity between cytochrome a and CuB is implied. The data are consistent with the hypothesis that oxidation of cytochrome a raises the midpoint potential of CuB by 55 +/- 10 mV.

Proton nuclear magnetic resonance characterization of the oxidized intermediates of cytochrome c peroxidase.

Oxidation of cytochrome c peroxidase with hydrogen peroxide to form the initial oxidized intermediate, cytochrome c peroxidase compound I, drastically alters the proton hyperfine nmr spectrum. In contrast to studies of horseradish peroxidase, where the spectrum of horseradish peroxidase compound I is similar to that of the native protein, cytochrome c peroxidase compound I exhibits only broad resonances near 17 and 30 ppm from 2,2-dimethyl-2-silapentane-5-sulfonate. No unique resonances attributable to cytochrome c peroxidase compound II could be identified. These results define the molecular conditions for which resolved hyperfine resonances of the iron(IV) states of heme proteins may be observed when the data presented here are compared with the data from horseradish peroxidase. Oxidation of cytochrome c peroxidase while it is complexed to ferricytochrome c reveals that the heme resonances of cytochrome c are not influenced by the oxidation state of cytochrome c peroxidase.

On the electron transfer between cytochrome c molecules as observed by nuclear magnetic resonance

Pulsed Fourier transform NMR studies on mixed solutions of the two oxidation states of cytochrome c reveal that the rate of exchange of oxidation states by electron transfer is sensitive to the conformation of the protein molecules. The electron transfer process in vitro is found to arise from binary collisions. At low ionic strengths, the rate is affected by the electrostatic charge on the protein molecules.

Electron Paramagnetic Resonance of Heme at Intermediate Oxidation States of Cytochrome c Oxidase

Electron Paramagnetic Resonance of Heme at Intermediate Oxidation States of Cytochrome <i>c</i> Oxidase

Electron paramagnetic resonance of heme at intermediate oxidation states of cytochrome c oxidase.

In past research on cytochrome c oxidase the only parameters measurable without (lestruction of the enzyme have been 02 uptake and substrate oxidation and the ()taical absorptions in the a and Soret region. To these have been added in recent years the electron paramagnetic resonance (EPR) absorption of the copper component, 2 the near infrared absorption at 830 mt,'-7 and CO binding capacity.8-'2 We wish to report here on three new, independently measurable parameters, viz., EPR absorptions due to heme components of the enzyme. EPR spectra showing weak heme absorption lines in oxidized'3 cytochrome c oxidase have been published previously. A line at g = 4.3,14 15 typical for asymmetric high-spin complexes of Fe(IJJ), and lines at g = 1.5 and g 31 characteristic for low-spin Fe(III) were detected. Cytochrome a3, thought to be a constituent of cytochrome c oxidase, is considered to be, at least partially, a high-spin compound. 14 For high-spin heme compounds an unusual and characteristic EPR absorption with q1 = 6, gql = 2 is well known.'6 It was therefore surprising that no such absorption was seen in cytochrome c oxidase. We have now found that very strong absorptions of this type are readily seen on partial reduction of the enzyme. In additon an EPR signal, typical for low-spin Fe(III), but different from the low-spin signal present in oxidized cytochrome c oxidase, emerges on partial reduction. Methods.--Cytochrome c oxidase was prepared according to Fowler et al.'7 It was titratedl anaerobically with diphosphopyridine nucleotide, reduced form (DPNH) in the presence of phenazine methosulfate (PMS) by the method of van Gelder.8 In an analogous fashion reduced cytochrome c or soliddithionite diluted with solid KCl were used as titrants. EPR spectroscopy and rapid mixing and freezing were carried out as described.20 The quantitative determination of the high-spin heme signal was based on a comparison with ferrimyoglobin fluoride and that of the low-spin signals on ferrimyoglobin azide. Since only parts of the cytochrome c oxidase EPR spectra are free of interference from other signals, it was assumed that the distribution of intensities over the different lines of each spectrum is the same as in the myoglobin derivatives. For both types of signals, therefore, only the low field lines were compared, which comprise 20 per cent of the total intensity for the high-spin signals and 7.5 per cent for the low-spin signals. These approximations may involve appreciable error, particularly with broad signals. This should be kept in mind when considering the meaning of plots such as that of Figure 2. Results.-Figure 1 shows a comparison of the signals obtained on partial reduction (DPNH) with those of oxidized cytochrome c oxidase. Qualitatively the same observations were made with either reduced cytochrome c with or without ascorbate, or dithionite as reductant. The upper curve corresponds to the oxidized state, the