Unusual redox behaviour of cytochrome b-561 from bovine chromaffin granule membranes

(1) Redox titrations of cytochrome b-561 have been performed with the purified cytochrome and with intact and detergent-solubilized chromaffin-granule membranes. (2) The midpoint redox potential of the cytochrome is 100–130 mV; this depends upon the composition of the buffer, but is independent of pH in the range 5.5–7.5; partial proteolysis of the cytochrome raises the midpoint potential to 160 mV. (3) The Nernst plots of titration data have slopes of 75–115 mV, and are in some cases sigmoid in shape. This may be explained by negative cooperativity during redox transitions in oligomeric cytochrome b-561. (4) Measurements of the haem and cytochrome content of chromaffin granule membrane suggest a haem content of 1 mol/mol protein. (5) Chemical crosslinking of cytochrome b-561 suggests that it may exist as an oligomer of 4–6 polypeptide chains within the chromaffin granule membrane. Aggregation of purified cytochrome b-561 was shown by gel filtration studies and by immunological methods in SDS-polyacrylamide gels. Studies of the molecular weight of the aggregates suggest that the monomer has a molecular weight close to 22 000, but migrates anomalously slowly during electrophoresis.     

The asymmetric orientation of cytochrome b561 in bovine chromaffin granule membranes

The topological arrangement of cytochrome b561 in the bovine adrenal medullary chromaffin granule membrane was investigated by radiolabeling and immunoprecipitation techniques using antibody raised against the purified cytochrome. The first labeling procedure involved a membrane-permeable amino group labeling reagent, ethyl acetimidate, and two membrane-nonpermeable amino group labeling reagents, isethionyl acetimidate and trinitrobenzenesulfonic acid. The second radiolabeling procedure involved lactoperoxidase-catalyzed iodination of the exposed tyrosines on the membrane-bound proteins. The labeled cytochrome b561 was isolated by immunoprecipitating detergent extracts of treated membranes, followed by electrophoresis of the precipitated cytochrome in polyacrylamide-dodecyl sulfate. From the analysis of both labeling techniques, cytochrome b561 appeared to be a transmembrane protein and a major portion of this protein was cytoplasmically exposed.     

Characterization by EPR spectroscopy of cytochrome b-562 isolated from the cytochrome b-c1 complex of Rhodopseudomonas sphaeroides R-26

The EPR spectra of cytochrome b-562 isolated from the cytochrome b-c1 complex of Rhodopseudomonas sphaeroides were measured at liquid helium temperature. The purified cytochrome b-562 gives a high spin signal at g = 6.0. Anaerobic titration of this signal confirmed the presence of two redox components with Em = 40 and -110 mV at pH 7.5. These values are consistent with the published ones, Em = 55 and -100 mV at pH 7.0, that were optically estimated for the same type of preparation (Iba et al. (1985) FEBS Lett. 183, 151-154). The power saturation behavior of the g = 6.0 signal at different redox potentials indicated a direct spin-spin interaction between these two redox centers.     

Oxido-reductive titrations of cytochrome c oxidase followed by EPR spectroscopy.

Experiments are described on oxido-reductive titrations of cytochrome c oxidase as followed by low-temperature EPR and reflectance spectroscopy. The reductants were cytochrome c or NADH and the oxidant ferricyanide. Experiments were conducted in the presence and absence of either cytochrome c or carbon monoxide, or both. An attempt is made to provide a complete quantitative balance of the changes observed in the major EPR signals. During reduction, the maximal quantity of heme represented in the high-spin ferric heme signals (g approximately 6; 2) is 25% of the total heme present, and during reoxidation 30%. With NADH reduction there is little difference between the pattern of disappearance of the low-spin ferric heme signals in the absence or presence of cytochrome c. The copper and high-spin heme signals, however, disappear at higher titrant concentrations in the presence of cytochrome c than in its absence. In these titrations, as well as in those with ferrocytochrome c, the quantitative balance indicates that, in addition to EPR-detectable components, EPR-undetectable components are also reduced, increasingly so at higher titrant concentrations. The quantity of EPR-undectable components reduced appears to be inverely related to pH. A similar inverse relationship exists between pH and appearance of high-spin signals during yhe titration. At pH 9.3 the quantity of heme represented in the high-spin signals is less than 5%, whereas it approximately doubles from pH 7.4 to pH 6.1. In the presence of CO less of the low-spin heme and copper signals disappears for the same quantity of titrant consumed, again implying reduction of EPR undetectable components. At least one of these components is represented in a broad absorption band centered at 655 nm. The stoichiometry observed on reoxidation, particularly in the presence of CO, is not compatible with the notion that the copper signal represents 100% of the active copper of the enzyme as a pair of interacting copper atoms.     

Purification of cytochrome B-561 from chromaffin granules and its physico-chemical properties

A soft method of purification of cytochrome-561 from the membranes of chromaffin granules has been developed. It permits isolating a protein in its natural microsurroundings, i.e. a complex with lipids, provided that a buffer with high ionic force is used without a detergent. This method helps obtaining an electrophoretically homogeneous preparation as a high-molecular lipoprotein hexamer whose molecular weight is about 400 kDa. Basic physicochemical parameters of this preparation (subunit composition, content and composition of lipids, heme content, spectra of optical absorption of the oxidized and reduced forms) are determined. Possible presence of two forms of cytochrome b-561 in the chromaffin granules is discussed.     

Properties of two distinct heme centers of cytochrome b561 from bovine chromaffin vesicles studied by EPR, resonance Raman, and ascorbate reduction assay

Cytochrome b(561) from bovine adrenal chromaffin vesicles contains two hemes b with different midpoint potentials (+150 and +60 mV) and participates in transmembrane electron transport from extravesicular ascorbate to an intravesicular monooxygenase, dopamine beta-hydroxylase. Treatment of oxidized cytochrome b(561) with diethylpyrocarbonate caused a downshift of midpoint potential for the lower component, and this shift was prevented by the presence of ascorbate during the treatment. Present EPR analyses showed that, upon the treatment, the g(z) = 3.69 heme species was converted to a non-ascorbate-reducible form, although its g(z)-value showed no appreciable change. The treatment had no effect on the other heme (the g(z) = 3.13 species). Raman data indicated that the two heme b centers adopt a six-coordinated low-spin state, in both the reduced and oxidized forms. There was no significant effect of diethylpyrocarbonate-treatment on the Raman spectra of either form, but the reducibility by ascorbate differed significantly between the two hemes upon the treatment. The addition of ferrocyanide enhanced both the reduction rate and final reduction level of the diethylpyrocarbonate-treated cytochrome b(561) when ascorbate was used as a reductant. This observation suggests that ferrocyanide scavenges monodehydroascorbate radicals produced by the univalent oxidation of ascorbate and, thereby, increases both the reduction rate and the final reduction level of the heme center on the intravesicular side of the diethylpyrocarbonate-treated cytochrome. These results further clarify the physiological role of this heme center as the electron donor to the monodehydroascorbate radical.     

Isolation of a membrane protein by chromatofocusing: Cytochrome b-561 of the adrenal chromaffin granule

Chromatofocusing, a form of ion-exchange chromatography in which proteins are separated on the basis of their differing isoelectric points, has been adapted for use with membrane proteins, solubilized by the non-ionic detergent Nonidet P-40. Using a two-step detergent extraction followed by chromatofocusing under high pressure, the highly hydrophobic protein cytochrome b-561 was isolated from chromaffin granule membranes and purified to near homogeneity in a functionally active form, in less than 5 h. Chromatofocusing conditions were optimized empirically since the behaviour of the chromaffin granule membrane proteins conformed less to the theory than that of soluble proteins, and the various factors affecting yield and resolution are discussed. The speed, high resolution and focusing effect could make this method particularly suitable for rapid isolation in a functionally active form of the many membrane proteins that are unstable in dilute solution and when removed from their lipid environment.     

Components of cytochrome c oxidase detectable by EPR spectroscopy

A procedure for the preparation from frozen beef heart mitochondria of cytochrome c oxidase (EC 1.9.3.1) of high heme ( 14 μmoles/mg protein) and low extraneous copper ( 1.1 atoms Cu/mole heme) and low lipid ( 0.05 g phospholipid/g protein) content is described. EPR signals observed with the enzyme between 6 and 100 °K at various states of oxidation and at different conditions of pH and presence of solutes are described in detail. The quantities of paramagnetic species represented by these signals are estimated. Under no conditions does the sum of the EPR detectable species represent more than approx. 50% of the potentially paramagnetic components of the enzyme. Comparisons are made to the corresponding signals as observed in whole tissue, mitochondria and submitochondrial particles from a number of species. The assignment of the observed signals to known components of cytochrome c oxidase is discussed briefly.     

pH-induced alteration and oxidative destruction of heme in purified chromaffin granule cytochrome b(561): implications for the oxidative stress in catecholaminergic neurons

The transmembrane hemoprotein, cytochrome b(561) (b(561)), in the neuroendocrine secretory vesicles is shown to shuttle electrons from the cytosolic ascorbate (Asc) to the intravesicular matrix to provide reducing equivalents for the dopamine beta-monooxygenase (DbetaM) reaction. Intravesicular Asc may also play a role in relieving catecholamine-induced oxidative stress in catecholaminergic neurons. In the present study, we have examined the alteration of purified oxidized b(561) (b(561,ox)) under mild alkaline conditions to probe the structural and functional characteristics of the protein, using UV-vis and EPR spectroscopic and kinetic techniques. Our results show that low spin heme in oxidized b(561) (b(561,ox)) readily transforms to an altered high spin form and then slowly to an Asc nonreducible form, in a pH-, temperature-, and time-dependent manner, which can be described by single-exponential rate equations, A(t) = A(o)(1- e (-kt)) and A(t) = A(o)e(-kt), respectively. More than half of the Asc nonreducible altered b(561) could be converted back to the native b(561) by pH adjustment followed by dithionite reduction, suggesting the reversibility of the process. The heme center of the transformed Asc nonreducible protein is completely bleached instantaneously by dithionite in the presence of atmospheric oxygen, which appears to be mediated by molecular oxygen and/or hydrogen peroxide. These results demonstrate that the heme centers of the protein are susceptible to the pH-induced alteration and oxidative destruction, raising some questions regarding the proposed one alkaline labile, two-heme model of b(561) [Tsubaki, M.; Nakayama, M.; Okuyama, E.; Ichikawa, Y. (1997) J. Biol. Chem. 272, 23206-23210]. The pH-induced alteration and the destruction of heme under oxidative conditions may play a significant role in the amplification of oxidative stress in catecholaminergic neurons.     

Oxido-reductive titrations of cytochrome c oxidase followed by EPR spectroscopy

Experiments are described on oxido-reductive titrations of cytochrome c oxidase as followed by low-temperature EPR and reflectance spectroscopy. The reductants were cytochrome c or NADH and the oxidant ferricyanide. Experiments were conducted in the presence and absence of either cytochrome c or carbon monoxide, or both. An attempt is made to provide a complete quantitative balance of the changes observed in the major EPR signals. During reduction, the maximal quantity of heme represented in the high-spin ferric heme signals (g ～ 6; 2) is 25% of the total heme present, and during reoxidation 30%. With NADH reduction there is little difference between the pattern of disappearance of the low-spin ferric heme signals in the absence or presence of cytochrome c. The copper and high-spin heme signals, however, disappear at higher titrant concentrations in the presence of cytochrome c than in its absence. In these titrations, as well as in those with ferrocytochrome c, the quantitative balance indicates that, in addition to EPR-detectable components, EPR-undetectable components are also reduced, increasingly so at higher titrant concentrations. The quantity of EPR-undetectable components reduced appears to be inversely related to pH. A similar inverse relationship exists between pH and appearance of high-spin signals during the titration. At pH 9.3 the quantity of heme represented in the high-spin signals is < 5%, whereas it approximately doubles from pH 7.4 to pH 6.1. In the presence of CO less of the low-spin heme and copper signals disappears for the same quantity of titrant consumed, again implying reduction of EPR undetectable components. At least one of these components is represented in a broad absorption band centered at 655 nm. The stoichiometry observed on reoxidation, particularly in the presence of CO, is not compatible with the notion that the copper signal represents 100% of the active copper of the enzyme as a pair of interacting copper atoms.     

Acidic copper-containing proteins--an intermediate link to the electron transfer stage from cytochrome b-561 to dopamine-beta-monooxygenase

The interaction of acidic copper-containing protein from the membranes of chromaffin granules has been investigated with cytochrome b-561 and dopamine-beta-monooxygenase from the same source. By the use of spectral and polarographic measurements it was demonstrated that the acidic copper-containing protein acts as an electron acceptor for cytochrome b-561 and as electron donor in the reactions, catalyzed by dopamine-beta-monooxygenase. According to the data obtained the possible function of the acidic copper-containing protein in vivo on the area of electron transfer chain between cytochrome b-561 and dopamine-beta-monooxygenase are discussed. The activation or inhibition of the electron transfer reactions by a variety of phospholipids, analogs of membrane lipids of chromaffin granules has been established. The experiments were performed in a model systems by the use of highly purified preparations of proteins and bilamellar liposomes and micelles, prepared from the corresponding phospholipids.     

Fluorescence spectroscopy studies of the association of membrane and soluble forms of chromaffin granule dopamine-beta-monooxygenase to bipolar phospholipid liposomes and micelles

The association of membrane and soluble forms of dopamine-beta-monooxygenase to liposomes and micelles made from phosphatidylcholine and lysophosphatidylcholine respectively has been studied using the fluorescence spectroscopy technique. As it was shown in our previous study these bipolar phospholipids activate the reaction catalyzed by the enzyme. Effects of pH and ionic strength on the association process were also studied, and efficiency of the association for apo- and holoenzyme was compared. The data obtained demonstrate that electrostatic attraction is involved in the association process. It was also shown that the membrane dopamine-beta-monooxygenase associated with phospholipid liposomes and micelles with higher efficiency than the soluble one did, which might be due to the involvement of the hydrophobic interactions in the association process. The results of the experiments also suggest that this process is specific and depends on the enzyme conformation, particularly on its quaternary structure. The participation of the hydrophobic peptide of the membrane dopamine-beta-monooxygenase in the formation and stabilization of the enzyme-phospholipid complex in vivo is proposed.     

Light-induced switching of HAMP domain conformation and dynamics revealed by time-resolved EPR spectroscopy

HAMP domains are widely abundant signaling modules. The putative mechanism of their function comprises switching between two distinct states. To unravel these conformational transitions, we apply site-directed spin labeling and time-resolved EPR spectroscopy to the phototactic receptor/transducer complex NpSRII/NpHtrII. We characterize the kinetic coupling of NpHtrII to NpSRII along with the activation period of the transducer and follow the transient conformational signal. The observed transient shift towards a more compact state of the HAMP domain upon light-activation agrees with structure-based calculations. It thereby validates the two modeled signaling states and integrates the domain’s dynamics into the current model.     

Stopped-flow analyses on the reaction of ascorbate with cytochrome b561 purified from bovine chromaffin vesicle membranes

Cytochrome b(561) in adrenal chromaffin vesicle membranes conveys electron equivalents from extravesicular ascorbate to the intravesicular monodehydroascorbate radical. We conducted a stopped-flow study on the reaction of ascorbate with purified cytochrome b(561) in the detergent-solubilized state for the first time. The time course of the reduction of oxidized cytochrome b(561) with ascorbate could not be fitted with a single exponential but with a linear combination of at least four exponential functions. This result is consistent with the notion that cytochrome b(561) contains two hemes b, each having a distinct redox potential and a function upon reactions with ascorbate and monodehydroascorbate radical. The fastest phase, which was assigned to the first one-electron donation from ascorbate to heme b on the extravesicular side, was further analyzed by transient phase kinetics employing a two-step bi-uni sequential ordered mechanism. The result showed K(s) = 2.2 mM for ascorbate at pH6.0. At a region below pH5.5, there was a significant lag before the reduction of hemes b occurred. This time lag was interpreted as due to a pH-dependent transient state before the first electron transfer to take place. The fastest phase was completely lost by N-carbethoxylation of heme-coordinating histidyl residues (His88 and His161) and Lys85 upon treatment with diethylpyrocarbonate. The presence of ascorbate during the treatment inhibited the N-carbethoxylation of the histidyl residues and, thereby, restored the final reduction level of hemes b. But the reduction rate was still only one-twentieth of the native form. This result suggested an important role of the conserved Lys85 for the interaction with ascorbate.     

Divalent cation-induced aggregation of chromaffin granule membranes.

Divalent cations induce the aggregation of chromaffin granule ghosts (CG membranes) at millimolar concentrations. Monovalent cations produce the same effect at 100-fold higher concentrations. The kinetics of the dimerization phase were followed by light-scattering changes observed in stopped-flow rapid mixing experiments. The rate constant for Ca2+-induced dimerization (kapp) is 0.86-1.0 x 10(9) M-1sec-1, based on the "molar" vesicle concentration. This value is close to the values predicted by theory for the case of diffusion-controlled reaction (7.02 x 10(9) M-1sec-1), indicating that there is no energy barrier to dimerization. Arrhenius plots between 10 degrees and 42 degrees C support this; the activation energy observed, +4.4 Kcal, is close to the value (4.6-4.8 Kcal) predicted for diffusion control according to theory. Artificial vesicles prepared from CG lipids were also found to have cation-induced aggregation, but the rates (values of kapp) were less than 1/100 as large as those with native CG membranes. Also, significant differences were found with respect to cation specificity. It is concluded that the slow rates are due to the low probability that the segments of membrane which approach will be matched in polar head group composition and disposition. Thus large numbers of approaches are necessary before matched segments come into aposition. The salient features of the chromaffin granule membrane aggregation mechanism are as follows: (a) In the absence of cations capable of shielding and binding, the membranes are held apart by electrostatic repulsion of their negatively charged surfaces. (b) The divalent and monovalent cation effects on aggregation are due to their ability to shield these charges, allowing a closer approach of the membrane surfaces. (c) The major determinants of the aggregation rates of CG membranes are proteins which protrude from the (phospholipid) surface of the membrane and serve as points of primary contact. Transmembrane contact between these proteins does not require full neutralization of the surface charge and surface potential arising from the negatively charged phospholipids. (d) After contact between proteins is established, the interaction between membranes can be strengthened through transmembrane hydrogen bonding of phosphatidyl ethanolamine polar head groups, divalent cation-mediated salt bridging, and segregation of phosphatidylcholine out of the region of contact.