Chromatography of oxidized and reduced cytochrome c on carboxymethylcellulose

1. Cytochrome c was isolated from horse heart by a chromatographic method. 2. Oxidized and reduced cytochrome c were chromatographed on CM-cellulose that was in equilibrium with several buffer systems of constant composition at pH values of 8.4, 6.75 and 4.9. 3. Separation was better at the higher pH values; the oxidized form was retarded more than twice as much as the reduced form, though they differed by only a single charge. 4. Self-competition between cytochrome molecules is suggested to account for the peak distortion observed at high loads (above 20mum protein concentration).     

Kinetic studies on oxidized and partially reduced cytochrome c oxidase.

1. Kinetic studies have been performed with beef-heart cytochrome c oxidase, with the enzyme either in its oxidized, resting state or pretreated anaerobically with different amounts of reduced cytochrome c. The techniques used for the study have been stopped-flow spectrophotometry and electron paramagnetic resonance (EPR) spectroscopy. 2. The results show that the one-electron equivalent-reduced enzyme rapidly oxidizes one further equivalent of aerobically or anaerobically added ferrocytochrome c, with a rate constant of 5 . 10(6) M-1 . s-1. 3. When an excess of ferrocytochrome c in the presence of oxygen is added to the one-electron-reduced enzyme, the same turnover rate is obtained as in experiments with the resting enzyme. 4. The one-electron equivalent-enzyme reacts with CO with a rate constant of 4 . 10(4) M-1 . s-1 to yield approx. 35% of the CO compound as compared with the reaction between the fully reduced enzyme and CO. 5. It is shown that on reduction the enzyme is converted into an active form, but it is concluded that the enzyme does not have to be fully reduced before it is catalytically active.     

Fast structural dynamics in reduced and oxidized cytochrome c

The sub‐nanosecond structural dynamics of reduced and oxidized cytochrome c were characterized. Dynamic properties of the protein backbone measured by amide ¹⁵N relaxation and side chains measured by the deuterium relaxation of methyl groups change little upon change in the redox state. These results imply that the solvent reorganization energy associated with electron transfer is small, consistent with previous theoretical analyses. The relative rigidity of both redox states also implies that dynamic relief of destructive electron transfer pathway interference is not operational in free cytochrome c.     

Potentiometric titration curves of oxidized and reduced horse heart cytochrome c

Potentiometric titration curves of oxidized and reduced horse heart cytochrome c in 0.15M KCl at 20°C have been obtained by timed titration (0.125–0.500 μmol/sec) from the isoionic points (pH 10.2–10.4) to pH 3 and back to the isoionic point. Computer-assisted (PROPHET) data acquisition and blank corrections give curves with good precision with a maximum standard deviation of 0.3 groups for an average error of 1%. The potentiometric titration curve of reduced cytochrome c is reversible within the precision of the method and for the pH range studied. The potentiometric curves for oxidized cytochrome c titrated upscale (pH 3–10) and downscale (pH 10–3) are not reversible. However, they show the same ionization behavior after the initial downscale titration. This is probably the result of a conformational change. Comparison of the data herein reported with the titration curves of oxidized cytochrome c already published by others indicates good agreement on the basis of a normalization of the concentration of protein or on the basis of 25 titrable groups between the acid end point and the isoionic pH. Titration of the 2 μmol imidazole in the upscale or downscale direction gives the correct analytical concentration and pK′ after correction for the solvent titration. Titration of reduced cytochrome c in the presence and absence of an additional equivalent of imidazole gave a difference titration curve, which indicates that a group on the protein shifts from pK′ 5.8 to pK′ 5.3 in the presence of imidazole. The pK′ of imidazole, in the presence of the protein, remains at a nearly normal value of 7.34.     

Surface properties of unsaturated non-oxidized and oxidized free fatty acids spread as monomolecular films at an argon/water interface

The interfacial properties of monomolecular films of stearic acid (SA) oleic acid (OA), linoleic acid (LA), ricinoleic acid (RA), 13(S)-hydroperoxyoctadeca-9Z,11E-dienoic acid (13-HPODE) and 13(S)-hydroxyoctadeca-9Z,11E-dienoic acid (13-HODE) were studied by recording the changes occurring in response to monomolecular film compression in their surface pressure and surface potential at the argon/water interface. The oxidized free fatty acids are more expanded than the parent non-oxidized free fatty acids, reflecting a higher hydrophilic-lipophilic balance. The lift-off values of the molecular area of 13-HODE, 13-HPODE and RA were 68, 74 and 106 A2 molecule(-1), respectively, as compared to 47 and 40 A2 molecule(-1) in the case of LA and OA, respectively. Variations in the molecular orientation of free fatty acids can result in large changes in the dipole moment which are not accompanied by appreciable changes in the surface pressure. In the case of the oxidized free fatty acids, the spontaneous desorption into the aqueous phase was found to increase at increasing surface pressures. The desorption rates of OA and LA increased dramatically in the presence of beta-cyclodextrin (beta-CD); whereas the presence of beta-CD only slightly increased the desorption rates of the oxidized free fatty acids.     

The reaction between reduced azurin and oxidized cytochrome c peroxidase from Pseudomonas aeruginosa

The reaction between ferric Pseudomonas cytochrome c peroxidase and reduced azurin was investigated by static titration, rapid scan, and stopped flow techniques as well as circular dichroism measurements. Kinetics of the reduction of the enzyme under pseudo-first order conditions reveals a biphasic logarithmic curve indicating that the reaction between enzyme and azurin is complex and comprises of two reactions, one rapid and a slower one. The relative portion of the fast phase from the overall reaction increases with increasing azurin concentration. Kinetic results can be explained by postulating the presence of two different enzyme forms which are slowly interconvertible. Both enzymatic forms form a complex with reduced azurin. The electron transfer between proteins occurs within the molecular complex of azurin and the peroxidase.     

Hydrogen-isotope exchange of oxidized and reduced cytochrome c. A comparison of mass spectrometry and infrared methods.

Hydrogen-deuterium exchange in 2H20 solutions of the two redox states of horse heart cytochrome c was investigated at 20 degrees C, pH 7, by mass spectrometry and infrared spectroscopy. Mass spectrometry indicates that ferricytochrome has 20 hydrogens unexchanged after 24 h, 28 hydrogens exchanging between 10 min and 24 h, and 156 hydrogens exchanging within 10 min; comparative values for ferrocytochrome are 45, 19 and 140. The displacement of the exchange curves obtained by infrared corresponds to 8 to 9 peptide hydrogens. These combined methods show many non-peptide hydrogens exchanging rapidly (87 and 79 for ferricytochrome c and ferrocytochrome c respectively), whereas others, probably buried inside the molecule and involved in hydrogen bonds, are not exchanged, even after 24 h (14 and 30 hydrogens respectively, which is relatively large for a small protein). Infrared results are given in terms of changes of standard free energy for the transconformational reaction which exposes the peptide hydrogens to solvent: in ferricytochrome c and ferrycoytochrome c, 30% and 40% respectively of the peptide hydrogens are protected by conformational transitions stabilized by more than 5 kcal/mol (21 kJ/mol), which implies a large increase in rigidity for the reduced form.     

Different types of interaction of oxidized and reduced cytochrome c with phospholipid vesicles]

The binding constants of ferri- and ferrocytochrome c interactions with phosphatidylcholine or cardiolipin-containing vesicules were determined. It was found that affinity of ferricytochrome c to phospholipids is one order of magnitude higher that of ferrocytochrome c. A comparative investigation of circular dichroism spectra of free and phospholipid-bound ferri- and ferrocytochrome c was undertaken and it was shown that alpha-helix content of free ferrocytochrome c is higher than that of ferricytochrome c. The formation of ferricytochrome c containing lipoprotein complex led to decrease of alpha-helix content of the protein. In the case of ferrocytochrome c on the other hand interaction with phospholipids did not cause any changes in alpha-helix content. Distribution of ferri- and ferrocytochrome c in different two-phase systems consisting of dextran and polyethylenglycol or dextran and polyethylenglvcol-palmitate was also studied. A comparison of distribution constants shows that higher alpha-helix content of ferrocytochrome c results in the formation of hydrophobic clusters in the protein molecules. In previous communications it was reported that binding of ferrocytochrome c to phospholipids is determined by hydrophobic interactions while in the case of ferricytochrome c the interactions with phospholipids are mainly electrostatic. On the basis of the results obtained in this work it is supposed that it is hydrophobic clusters which determine the binding of ferrocytochrome c to phospholipid membranes.     

Small substrates and cytochrome c are oxidized at different sites of cytochrome c peroxidase.

Modeling studies suggest that electrons are transferred from cytochrome c to cytochrome c peroxidase (CcP) with cytochrome c predominantly bound at a site facing the gamma-meso edge of the CcP prosthetic heme group (Poulos, T.L., and Kraut, J. (1980) J. Biol. Chem. 255, 10322-10330). As shown here, guaiacol and ferrocyanide are oxidized at a different site of CcP. Thus, the oxidations of cytochrome c and guaiacol are differentially inactivated by phenylhydrazine and sodium azide. The loss of guaiacol oxidation activity correlates with covalent binding of 1 equivalent of [14C]phenylhydrazine to the protein, whereas the slower loss of cytochrome c activity correlates with the appearance of a 428-nm absorbance maximum attributed to the formation of a sigma-phenyl-iron heme complex. The delta-meso-phenyl and 8-hydroxymethyl derivatives of heme are formed as minor products. Catalytic oxidation of azide to the azidyl radical results in inactivation of CcP and formation of delta-meso-azidoheme. Reconstitution of apo-CcP with delta-meso-azido-, -ethyl-, and -(2-phenylethyl)heme yields holoproteins that give compound I species with H2O2 and exhibit 80, 59, and 31%, respectively, of the control kcat value for cytochrome c oxidation but little or no guaiacol or ferrocyanide oxidizing activity. Conversely, CcP reconstituted with gamma-meso-ethylheme is fully active in the oxidation of guaiacol and ferrocyanide but only retains 27% of the cytochrome c oxidizing activity. These results indicate that guaiacol and ferrocyanide are primarily oxidized near the delta-meso-heme edge rather than, like cytochrome c, at a surface site facing the gamma-meso edge.     

Molecular dynamics study of Desulfovibrio africanus cytochrome c3 in oxidized and reduced forms

A 5-ns molecular dynamics study of a tetraheme cytochrome in fully oxidized and reduced forms was performed using the CHARMM molecular modeling program, with explicit water molecules, Langevin dynamics thermalization, Particle Mesh Ewald long-range electrostatics, and quantum mechanical determination of heme partial charges. The simulations used, as starting points, crystallographic structures of the oxidized and reduced forms of the acidic cytochrome c(3) from Desulfovibrio africanus obtained at pH 5.6. In this paper we also report structures for the two forms obtained at pH 8. In contrast to previous cytochrome c(3) dynamics simulations, our model is stable. The simulation structures agree reasonably well with the crystallographic ones, but our models show higher flexibility and the water molecules are more labile. We have compared in detail the differences between the simulated and experimental structures of the two redox states and observe that the hydration structure is highly dependent on the redox state. We have also analyzed the interaction energy terms between the hemes, the protein residues, and water. The direct electrostatic interaction between hemes is weak and nearly insensitive to the redox state, but the remaining terms are large and contribute in a complex way to the overall potential energy differences that we see between the redox states.     

Calorimetric determination of the enthalpy of ionization of oxidized and reduced horse heart cytochrome c

The heats of ionization of protons, ΔH_i, of oxidized and reduced horse heart cytochrome c in 0.15M KCl at 20°C were determined using a titration calorimeter which simultaneously afforded the potentiometric titration curve. Reproducibility of the thermal titrations is within 2%, and evaluation of the heats observed after the heat loss corrections is estimated to be within 5%. A single titration of oxidized cytochrome c from pH 11 to 3 is in excellent agreement with the thermal titration of this protein obtained with flow calorimetry. The thermal titration, however, is not reversible, due in part to the loss of titratable group(s) in this pH region and to the heat contribution of the acid and alkaline conformational changes which occur. Although of lesser magnitude, the reduced form also indicates similar thermal transitions. These differences are due solely to conformational contributions to the thermal process, since the potentiometric curves are reversible. The nature of the irreversibility for oxidized cytochrome c appears to involve the loss of a group with pK′ 8.9 and the shift of two groups from pK′ 5.6 to 4.8. Thermal difference curves for this process indicate that heats of ?7.8 and ?24.1 kcal/mol are liberated which are centered at pH 9.3 and 3.9, respectively.     

High-resolution structures of the oxidized and reduced states of cytochrome c554 from Nitrosomonas europaea

Cytochrome c554 (cyt c554) is a tetra-heme cytochrome involved in the oxidation of NH3 by Nitrosomonas europaea. The X-ray crystal structures of both the oxidized and dithionite-reduced states of cyt c554 in a new, rhombohedral crystal form have been solved by molecular replacement, at 1.6 A and 1.8 A resolution, respectively. Upon reduction, the conformation of the polypeptide chain changes between residues 175 and 179, which are adjacent to hemes III and IV. Cyt c554 displays conserved heme-packing motifs that are present in other heme-containing proteins. Comparisons to hydroxylamine oxidoreductase, the electron donor to cyt c554, and cytochrome c nitrite reductase, an enzyme involved in nitrite ammonification, reveal substantial structural similarity in the polypeptide chain surrounding the heme core environment. The structural determinants of these heme-packing motifs extend to the buried water molecules that hydrogen bond to the histidine ligands to the heme iron. In the original structure determination of a tetragonal crystal form, a cis peptide bond between His129 and Phe130 was identified that appeared to be stabilized by crystal contacts. In the rhombohedral crystal form used in the present high-resolution structure determination, this peptide bond adopts the trans conformation, but with disallowed angles of phi and psi.     

A proton-NMR investigation of the fully reduced cytochrome c7 from Desulfuromonas acetoxidans. Comparison between the reduced and the oxidized forms.

The solution structure via 1H NMR of the fully reduced form of cytochrome c7 has been obtained. The protein sample was kept reduced by addition of catalytic amounts of Desulfovibrio gigas iron hydrogenase in H2 atmosphere after it had been checked that the presence of the hydrogenase did not affect the NMR spectrum. A final family of 35 conformers with rmsd values with respect to the mean structure of 8.7 +/- 1.5 nm and 12.4 +/- 1.3 nm for the backbone and heavy atoms, respectively, was obtained. A highly disordered loop involving residues 54-61 is present. If this loop is ignored, the rmsd values are 6.2 +/- 1.1 nm and 10.2 +/- 1.0 nm for the backbone and heavy atoms, respectively, which represent a reasonable resolution. The structure was analyzed and compared with the already available structure of the fully oxidized protein. Within the indetermination of the two solution structures, the result for the two redox forms is quite similar, confirming the special structural features of the three-heme cluster. A useful comparison can be made with the available crystal structures of cytochromes c3, which appear to be highly homologous except for the presence of a further heme. Finally, an analysis of the factors affecting the reduction potentials of the heme irons was performed, revealing the importance of net charges in differentiating the reduction potential when the other parameters are kept constant.     

The interactions of cytochrome c and porphyrin cytochrome c with cytochrome c oxidase. The resting, reduced and pulsed enzymes

Cytochrome c oxidase forms tight binding complexes with the cytochrome c analog, porphyrin cytochrome c. The behaviour of the reduced and pulsed forms of the oxidase with porphyrin cytochrome c have been followed as functions of ionic strength; this behaviour has been compared with that of the resting oxidase [Kornblatt, Hui Bon Hoa and English (1984) Biochemistry 23, 5906-5911]. All forms of the cytochrome oxidase studied bind one porphyrin cytochrome c per 'functional' cytochrome oxidase (two heme a); it appears as though porphyrin cytochrome c and cytochrome c compete for the same site on the oxidase. The resting enzyme binds cytochrome c 8 times more strongly than porphyrin cytochrome c; the reduced enzyme, in contrast, binds the two with almost equal affinity. In all three cases, resting, pulsed and reduced, the heme-to-porphyrin distance is estimated to be about 3 nm. The tight-binding complexes formed between cytochrome oxidase and porphyrin cytochrome c can be dissociated by salt. Debye-Hückel analysis of salt titrations indicate that the resting enzyme and the reduced enzyme are similar in that the product of the interaction charges on the two proteins is about -14. The product of the charges for the pulsed enzyme is -25, indicating that on average another positive and negative charge take part in the interaction of the two proteins. While there is one tight binding site for cytochrome c per two heme a, cytochrome c is able to 'communicate' with four heme a. In the absence of cytochrome c, electron transfer from tetramethylphenylenediamine to the oxidase to oxygen results in the conversion of the resting form to the 'oxygenated'; in the presence of cytochrome c, the same electron transfer results in the appearance of the 'pulsed' form. Cytochrome c titrations of the enzyme show that a ratio of only one cytochrome c to four heme a is sufficient to convert all the oxidase to the 'pulsed' form. Porphyrin cytochrome c, like cytochrome c, catalyzes the same conversion with the same stoichiometry. The binding data and salt effects indicate that major structural alterations occur in the oxidase as it is converted from the resting to the partially reduced and subsequently to the pulsed form.     

Differences in the solution structures of oxidized and reduced cytochrome c measured by small-angle X-ray scattering

While X-ray crystallographic data on cytochrome c show the reduced and oxidized forms to have very similar structures, there is a considerable body of data, mostly from solution studies, that indicates the reduced form is more stable and that the interior of the protein is less accessible to solvent in this state. These observations have led to the hypothesis that while the time-averaged structure is preserved between the two forms, the dynamics of the two forms are different. The oxidized form has been proposed to undergo more large-amplitude, low-frequency motions than the reduced form. The crystal structure data were derived from crystals grown in high salt concentrations, but the solution studies were done at relatively low ionic strength. Small-angle X-ray scattering has been used to examine the effects of the ionic strength and oxidation state on the solution structure of cytochrome c. We find that the radius of gyration and the maximum linear dimension of oxidized cytochrome c are significantly larger than those for reduced cytochrome c, in 5 mM phosphate buffer at pH 7.3, and further that this difference is suppressed by addition of 200 mM sodium chloride. We conclude that there is a real structural difference between the two forms at low ionic strength in solution and that this difference is likely to contribute to the observed differences in accessibility and compressibility.