Purification and properties of a cross-linked complex between cytochrome c and cytochrome c peroxidase.

Cytochrome c (horse heart) was covalently linked to yeast cytochrome c peroxidase by using the cleavable bifunctional reagent dithiobis-succinimidyl propionate in 5 mM-sodium phosphate buffer, pH 7.0. A cross-linked complex of molecular weight 48 000 was purified in approx. 10% yield from the reaction mixture, which contained 1 mol of cytochrome c and 1 mol of cytochrome c peroxidase/mol. Of the total 40 lysine residues, four to six were blocked by the cross-linking agent. Dithiobis-succinimidylpropionate can also cross-link cytochrome c to ovalbumin, but cytochrome c peroxidase is the preferred partner for cytochrome c in a mixture of the three proteins. The cytochrome c cross-linked to the peroxidase can be rapidly reduced by free cytochrome c-557 from Crithidia oncopelti, and the equilibrium obtained can be used to calculate a mid-point oxidation-reduction potential for the cross-linked cytochrome of 243 mV. Mitochondrial NADH-cytochrome c reductase will reduce the bound cytochrome only very slowly, but the rate of reduction by ascorbate at high ionic strength approaches that for free cytochrome c. Bound cytochrome c reduced by ascorbate can be re-oxidized within 10s by the associated peroxidase in the presence of equimolar H2O2. In the standard peroxidase assay the cross-linked complex shows 40% of the activity of the free peroxidase. Thus the intrinsic ability of each partner in the complex to take part in electron transfer is retained, but the stable association of the two proteins affects access of reductants.     

Site-directed chemical modification of horse cytochrome c results in changes in antigenicity due to local and long-range conformational perturbations

Comparative binding studies with peptide fragments of the whole antigen, or with evolutionarily related intact proteins with varying degrees of sequence homology, have been used extensively to map antigenic sites on proteins to the resolution of single amino acid residues. These methods are limited, however, since high affinity antibodies will often not react with peptides and evolutionarily related proteins are available for only a few antigens. In this study we use site-directed chemical modification of horse cytochrome c to identify residues involved in the binding sites of four monoclonal antibodies specific for this protein. Thus, we have N-formylated the single tryptophan found in horse cytochrome c at position 59 and N-carbethoxylated one of the histidyl residues, which was determined to be at position 26 by the analysis of proteolytic cleavage fragments of the modified protein using liquid secondary ion-mass spectrometry on triple quadropole or tandem quadropole Fourier transform instruments. We discuss the impact of these modifications on the antigenicity of horse cytochrome c with regard to the conformational perturbations introduced by such modifications and with reference to our previous studies on the binding sites of these antibodies using other methodologies (Jemmerson, R., and Paterson, Y. (1986) BioTechniques 4, 18-31).     

Folding of thermolysin fragments. Correlation between conformational stability and antigenicity of carboxyl-terminal fragments

Circular dichroism (CD) and immunochemical measurements have been used to examine conformational properties of COOH-terminal fragments 121-316, 206-316 and 225(226)-316 of thermolysin, and to compare these properties to those of native thermolysin and thermolysin S, the stable partially active two-fragment complex composed of fragments 5-224(225) and 225(226)-316. In aqueous solution at neutral pH, all the COOH-terminal fragments attain a native-like conformation, as judged both by the content of secondary structure deduced from far-ultraviolet CD spectra and by the recognition of rabbit polyclonal antibodies specific for the COOH-terminal region in native thermolysin. The three fragments showed reversible cooperative unfolding transitions mediated by both heat and guanidine hydrochloride (Gdn X HCl). The phase transition curves were analyzed for Tm (temperature of half-denaturation) and Gibbs free energies (delta GD) of unfolding from native to denatured state. The observed order of thermal stability is 225(226)-316 less than or equal to 206-316 less than 121-316 less than thermolysin S less than thermolysin. The ranking of delta GD values for the three fragments correlates with the size of each fragment. Competitive binding studies by radioimmunoassay using 14C-labeled thermolysin and affinity purified antibodies specific for native antigenic determinants in segment 206-316 of native thermolysin indicate that the COOH-terminal fragments adopt native-like conformations which are in equilibrium with non-native conformations. These equilibria are shifted towards the native state as the fragment size increases from 225(226)-316, to 206-316, to 121-316. Fragment 225(226)-316, when combined with fragment 5-224(225) in the thermolysin S complex, adopts a more stable native-like conformation and becomes much more antigenic. It has been shown that the degree of antigenicity of COOH-terminal fragments towards thermolysin antibodies correlates directly with their conformational stability. The results of this study are discussed in relation to the recently proposed correlation between antigenicity and segmental mobility of globular proteins.     

Zinc cytochrome c fluorescence as a probe for conformational changes in cytochrome c oxidase.

Zinc cytochrome c forms tight 1:1 complexes with a variety of derivatives of cytochrome c oxidase. On complex-formation the fluorescence of zinc cytochrome c is diminished. Titrations of zinc cytochrome c with cytochrome c oxidase, followed through the fluorescence emission of the former, have yielded both binding constants (K approximately 7 x 10(6) M-1 for the fully oxidized and 2 x 10(7) M-1 for the fully reduced enzyme) and distance information. Comparison of steady-state measurements obtained by absorbance and fluorescence spectroscopy in the presence and in the absence of cyanide show that it is the reduction of cytochrome a and/or CuA that triggers a conformational change: this increases the zinc cytochrome c to acceptor (most probably cytochrome a itself) distance by some 0.5 nm. Ligand binding to the fully oxidized or fully reduced enzyme leaves the extent of fluorescence quenching unchanged, whereas binding of cyanide to the half-reduced enzyme (a2+CuA+CuB2+-CN(-)-a3(3+)) enhances fluorescence emission relative to that for the fully reduced enzyme, implying further relative movement of donor and acceptor.     

ATP induces a conformational change in lipid-bound cytochrome c

Resonance energy transfer studies using a pyrene-labeled phospholipid derivative 1-palmitoyl-2-[10-(pyren-1-yl)decanoyl]-sn-glycero-3-phosphoglycerol (donor) and the heme (acceptor) of cytochrome c (cyt c) have indicated that ATP causes changes in the conformation of the lipid-bound protein (Ryt?maa, M., Mustonen, P., and Kinnunen, P. K. J. (1992) J. Biol. Chem. 267, 22243-22248). Accordingly, after binding cyt c via its so called C-site to neat phosphatidylglycerol liposomes (mole fraction of PG = 1.0) has commenced, further quenching of donor fluorescence is caused by ATP, saturating at 2 mm nucleotide. ATP-induced conformational changes in liposome-associated cyt c could be directly demonstrated by CD in the Soret band region (380-460 nm). The latter data were further supported by time-resolved spectroscopy using the fluorescent cyt c analog with a Zn(2+)-substituted heme moiety. A high affinity ATP-binding site has been demonstrated in cyt c (Craig, D. B., and Wallace, C. J. A. (1993) Protein Sci. 2, 966-976) that is compromised by replacing the invariant Arg(91) to norleucine. Although no major effects on conformation and function of cyt c were concluded due to the modification, a significantly reduced effect by ATP on the lipid-bound [Nle(91)]cyt c was evident, implying that this modulation is mediated via the Arg(91)-containing binding site.     

A covalent complex between horse heart cytochrome c and yeast cytochrome c peroxidase: kinetic properties

The kinetic properties of a 1:1 covalent complex between horse-heart cytochrome c and yeast cytochrome c peroxidase (ferrocytochrome-c:hydrogen-peroxide oxidoreductase, EC 1.11.1.5) have been investigated by transient-state and steady-state kinetic techniques. Evidence for heterogeneity in the complex is presented. About 50% of the complex reacts with hydrogen peroxide with a rate 20–40% faster than that of native enzyme; 20% of the complex exists in a conformation which does not react with hydrogen peroxide but converts to the reactive form at a rate of 20 ± 5 s⁻¹; 30% of the complex does not react with hydrogen peroxide to form the oxidized enzyme intermediate, cytochrome c peroxidase Compound I. Intramolecular electron transfer between covalently bound ferrocytochrome c and an oxidized site in cytochrome c peroxidase Compound I is too fast to measure, but a lower limit of 600 s⁻¹ can be estimated at 5°C in a 10 mM potassium phosphate buffer at pH 7.5. Free ferrocytochrome c reduces cytochrome c peroxidase Compound I covalently bound to ferricytochrome c at a rate 10⁻⁴ to 10⁻⁵-times slower than for free Compound I. The transient-state ferrocytochrome c reduction rates of Compound I covalently linked to ferricytochrome c are about 70-times too slow to account for the steady-state catalytic properties of the 1:! covalent complex. This indicates that hydrogen peroxide can interact with the 1:1 complex at sites other than the heme of cytochrome c peroxidase, generating additional species capable of oxidizing free ferrocytochrome c.     

Oxidation of cytochrome c by cytochrome c oxidase: spectroscopic binding studies and steady-state kinetics support a conformational transition mechanism

The long-known biphasic response of cytochrome c oxidase to the concentration of cytochrome c has been explained, alternatively, by the presence of a catalytic and a regulatory site on the oxidase, by negative cooperativity between adjacent active sites in dimeric oxidase, or by a transition of the enzyme molecule between different conformational states. The three mechanistic hypotheses allow testable predictions about the relationship between substrate binding and steady-state kinetics catalyzed by the monomeric and dimeric (or oligomeric) enzyme. We have tested these predictions on monomeric, dimeric, and oligomeric beef heart oxidase and on monomeric oxidase from Paracoccus denitrificans. The aggregation state of the oxidase was evaluated from the sedimentation equilibrium in the ultracentrifuge and by gel chromatography. The binding of cytochrome c to cytochrome c oxidase was measured by spectrophotometric titration of cytochrome c oxidase with cytochrome c. The procedure makes use of a small perturbation in the Soret band of the absorption spectrum of the cytochrome c-cytochrome c oxidase complex. The steady-state oxidation of cytochrome c was followed spectroscopically by an automated assay procedure, and the kinetic parameters were deduced by numerical analysis of several hundred initial rate assays in the substrate concentration range 0.15-30 microM. The following results were obtained: (1) The kinetics of cytochrome c oxidation are always biphasic at low ionic strength, independent of the aggregation state of the enzyme. (2) The kinetics become apparently monophasic at ionic strengths above 100 mM or at slightly acidic pH values.(ABSTRACT TRUNCATED AT 250 WORDS)     

A spin label study of conformational changes in cytochrome c

Spin-labeled pig heart cytochromes c singly modified at Met-65, Tyr-74 and at one of the lysine residues, Lys-72 or Lys-73, were investigated by the ESR method under conditions of different ligand and redox states of the heme and at various pH values. Replacement of Met-80 by the external ligand, cyanide, was shown to produce a sharp increase in the mobility of all the three bound labels while reduction of the spin-labeled ferricytochromes c did not cause any marked changes in their ESR spectra. In the pH range 6-13, two conformational transitions in ferricytochrome c were observed which preceded its alkaline denaturation: the first with pK 9.3 registered by the spin label at the Met-65 position, and the second with pK 11.1 registered by the labels bound to Tyr-74 and Lys-72(73). The conformational changes in the 'left-hand part' of ferricytochrome c are most probably induced in both cases by the exchange of internal protein ligands at the sixth coordination site of the heme.     

Spectroscopic analysis of the interaction between cytochrome c and cytochrome c oxidase

Complex formation between cytochrome c oxidase and cytochrome c perturbs the optical absorption spectrum of heme c and heme a in the region of the alpha-, beta, and gamma-bands. The perturbations have been used to titrate cytochrome c oxidase with cytochrome c. A stoichiometry of one molecule of cytochrome c bound per molecule of cytochrome c oxidase is obtained (1 heme c per heme aa3). In contrast, a stoichiometry of 2:1 was found earlier using a gel-filtration method (Rieder, R., and Bosshard, H.R. (1978) J. Biol. Chem. 253, 6045-6053). From the result of the spectrophotometric titration and from the wavelength position of the perturbation signals it is concluded that cytochrome c oxidase contains only a single binding site for cytochrome c which is close enough to heme a to function as an electron transfer site. The second site detected earlier by the gel-filtration method must be remote from this electron transfer site. Scatchard plots of the titration data are curvilinear, possibly indicating interactions between cytochrome c-binding sites on adjacent monomers of dimeric cytochrome c oxidase. The relationship between cytochrome c binding and the reaction of cytochrome c oxidase with ferrocytochrome c is discussed.     

Monoclonal antibodies as probes of conformational changes in protein-engineered cytochrome c

Determination of the nature of the antigen-antibody complex has always been the ultimate goal of three-dimensional epitope mapping studies. Various strategies for epitope mapping have been employed which include comparative binding studies with peptide fragments of antigens, binding studies with evolutionarily related proteins, chemical modifications of epitopes, and protection of epitopes from chemical modification or proteolysis by antibody shielding. In this study we report the use of protein engineering to modify residues in horse cytochrome c that are in or near the epitopes of four monoclonal antibodies specific for this protein. The results demonstrate not only that site-specific changes in the antigen binding site dramatically affect antibody binding, but, more importantly, that some of the site-specific changes cause local and long-range perturbations in structure that are detected by monoclonal antibody binding at other surfaces of the antigen. These findings emphasize the role of native conformation in the stabilization of the interaction between protein antigens and high affinity monoclonal antibodies. Furthermore, the results demonstrate that monoclonal antibodies are more sensitive probes of changes in conformation brought about by protein engineering than low resolution spectroscopic methods such as circular dichroism, where similar spectra are observed for all the analogues. These findings suggest a role for monoclonal antibodies in detecting conformational changes invoked by nonconservative amino acid substitutions or substitutions of evolutionarily conserved residues in protein-engineered or recombinant proteins.     

Electron transfer between cytochrome c2 and the tetraheme cytochrome c in Rhodopseudomonas viridis

Kinetics of electron transfer from soluble cytochrome c₂ to the tetraheme cytochrome c have been measured in isolated reaction centers and in membrane fragments of the photosynthetic purple bacterium Rhodopseudomonas viridis by time-resolved flash absorption spectroscopy. Absorbance changes kinetics in the region of cytochrome -bands (540–560 nm) were measured at 21 °C under redox conditions where the two high-potential hemes (c-559 and c-556) of the tetraheme cytochrome were chemically reduced. After flash excitation, the heme c-559 donates an electron to the special pair of bacteriochlorophylls and is then re-reduced by heme c-556. The data show that oxidized heme c-556 is subsequently re-reduced by electron transfer from reduced cytochrome c₂ present in the solution. The rate of this reaction has a non-linear dependence on the concentration of cytochrome c₂, suggesting a (minimal) two-step mechanism involving the f ormation of a complex between cytochrome c₂ and the reaction center, followed by intracomplex electron transfer. To explain the monophasic character of the reaction kinetics, we propose a collisional mechanism where the lifetime of the temporary complex is short compared to electron transfer. The limit of the halftime of the bimolecular process when extrapolated to high concentrations of cytochrome c₂ is 60 ± 20 s. There is a large ionic strength effect on the kinetics of electron transfer from cytochrome c₂ to heme c-556. The pseudofirst-order rate constant decreases from 1.1 × 10⁷ M^-1 s^-1 to 1.3 × 10⁶ M^-1 s^-1 when the ionic strength is increased from 1 to 1000 mM. The maximum rate (1.1 × 10⁷ M^-1 s^-1) was obtained at about 1 mM ionic strength. This dependence of the rate on ionic strength s uggests that attractive electrostatic interactions contribute to the binding of cytochrome c₂ with the tetraheme cytochrome. On the basis of our data and of previous molecular modelling, it is proposed that cytochrome c₂ docks close to the low-potential heme c-554 and reduces heme c-556 via c-554.     

Some properties of the redox components of cytochrome c oxidase and their interactions.

The electron paramagnetic resonance (epr) properties of cytochrome c oxidase have been examined with special attention to the effect of added ligands and of interactions between the redox components. The fully oxidized preparations have a very small g6 signal which increases greatly as the redox potential is made more negative, a process exactly paralleling the disappearance of the g3 signal. The potential for half appearance or disappearance (E_m), respectively, is 380 mV at pH 7.0 and 300 mV at pH 8.5. This identifies the changes as accompanying reduction of cytochrome a₃ because the E_m of the “invisible copper” is 340 mV and pH independent. Nitric oxide (NO) binds reduced cytochrome a₃ to form a paramagnetic species. This resulting epr signal is strongly dependent on the redox state of cytochrome a, another expression of heme-heme interaction in cytochrome oxidase. The NO compound is also unique in that under the appropriate conditions three of the four redox components (cytochrome a₃, cytochrome a, and the “visible” copper) are epr active. In potentiometric titrations in the presence of azide the formation of the azide compound responsible for the g2.9 signal appears to require reduction of both cytochrome a₃ and the “invisible copper.” An internal sulfur compound is present which, at alkaline pH values, can bind the heme responsible for the g6 signal and change it to a low-spin sulfur compound with a signal at approximately g2.6. Evidence is also presented for the cytochrome c oxidase in situ being an equilibrium mixture of two different conformational states.     

Reduction of CuA induces a conformational change in cytochrome c oxidase from Paracoccus denitrificans.

Cytochrome c oxidase (cytochrome aa3) from Paracoccus denitrificans contains a tightly bound manganese(II) ion, which responds to reduction of the enzyme by a change in its EPR signal (Seelig et al. (1981) Biochim. Biophys. Acta 636, 162-167). In this paper, the nature of this phenomenon is studied and the bound manganese is used as a reporter group to monitor a redox-linked conformational change in the protein. A reductive titration of the cyanide-inhibited enzyme shows that the change in the manganese EPR signal is associated with reduction of CuA. The change appears to reflect a rearrangement in the rhombic octahedral coordination environment of the central Mn2+ atom and is indicative of a redox-linked conformational transition in the enzyme. The manganese is likely to reside at the interface of subunits I and II, near the periplasmic side of the membrane. One of its ligands may be provided by the transmembrane segment X of subunit I, which has been suggested to contribute ligands to cytochrome a and CuB as well. Another manganese ligand is a water oxygen, as indicated by broadening of the manganese EPR signal in the presence of H2(17)O.     

The conformational states of oxidized and oxygenated beef-heart cytochrome c oxidase.

1. The "oxygenated" form of cytochrome has been generated by treatment of the enzyme with ascorbic acid. 2. "Oxygenated oxidase" so generated is stable over long periods (24 h). 3. Sedimentation velocity experiments have shown the "oxygenated" oxidase to be a less compact molecule than the oxidized.     

Physical and catalytic properties of a peroxidase derived from cytochrome c

Except for its redox properties, cytochrome c is an inert protein. However, dissociation of the bond between methionine-80 and the heme iron converts the cytochrome into a peroxidase. Dissociation is accomplished by subjecting the cytochrome to various conditions, including proteolysis and hydrogen peroxide (H(2)O(2))-mediated oxidation. In affected cells of various neurological diseases, including Parkinson's disease, cytochrome c is released from the mitochondrial membrane and enters the cytosol. In the cytosol cytochrome c is exposed to cellular proteases and to H(2)O(2) produced by dysfunctional mitochondria and activated microglial cells. These could promote the formation of the peroxidase form of cytochrome c. In this study we investigated the catalytic and cytolytic properties of the peroxidase form of cytochrome c. These properties are qualitatively similar to those of other heme-containing peroxidases. Dopamine as well as sulfhydryl group-containing metabolites, including reduced glutathione and coenzyme A, are readily oxidized in the presence of H(2)O(2). This peroxidase also has cytolytic properties similar to myeloperoxidase, lactoperoxidase, and horseradish peroxidase. Cytolysis is inhibited by various reducing agents, including dopamine. Our data show that the peroxidase form of cytochrome c has catalytic and cytolytic properties that could account for at least some of the damage that leads to neuronal death in the parkinsonian brain.