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E Margoliash

Publications and source records attributed to E Margoliash.

At least 73 records · Page 4Linked to original sources
PubMed1983

Control of the transfer of oxidizing equivalents between heme iron and free radical site in yeast cytochrome c peroxidase.

A procedure has been developed for obtaining yeast cytochrome c peroxidase with the heme iron in the Fe(IV) state, without the concomitant formation of the protein free radical that occurs in the ES compound resulting from the oxidation of ferric peroxidase with hydrogen peroxide. In this procedure, ferrous peroxidase, prepared either by photochemical reduction or by trapping the dithionite-reduced enzyme with carbon monoxide, is oxidized with a stoichiometric amount of hydrogen peroxide. The resulting Fe(IV) enzyme oxidizes ferrocyanide monophasically, with a rate constant of 4 x 10(3) M-1 S-1. The optical spectrum of the free radical was obtained as the difference between the spectra of the ES and Fe(IV) compounds. EPR spectra of ES compound prepared with [16O]-and [17O]hydrogen peroxide are identical, demonstrating that no fragment of the oxidant is associated with the free radical. The heme in the Fe(IV) enzyme is stable and does not oxidize the free radical site either intra- or intermolecularly. On the other hand, previous results from the presteady state kinetics of reduction and reductive titrations of the ES compound with ferrocyanide imply that the heme and free radical sites exchange oxidizing equivalents, in particular that the radical site once reduced can be reoxidized, either intra- or intermolecularly, by the ferryl heme. To resolve these contradictions, we propose a catalytic mechanism for cytochrome c peroxidase in which the radical site can exist in two conformations having very different reduction potentials and in which a significant flow of oxidizing equivalents between heme and free radical sites occurs only (i) during the hydrogen peroxide oxidation of the resting Fe(III) enzyme to form compound ES and (ii) within the initial transient intermediate formed upon the one-electron reduction of this oxidized product.

Cytochrome-c Peroxidase↗
PubMed1982

Kinetics and mechanism of the reduction of ferricytochrome c by the superoxide anion.

The temperature and pH dependence of the reaction of the superoxide radical anion with ferricytochrome c have been measured using the pulse-radiolysis technique. The temperature dependence of the reaction at low ionic strength yields an activation energy of 31 +/- 5 kJ/mol as compared to 14 +/- 3 kJ/mol for the reaction of CO2.(-) under the same conditions. The pH dependence fits the single pK'a of ferricytochrome c of 9.1. The bimolecular rate constant for the reaction of the superoxide anion with ferricytochrome c at pH 7.8, 21 +/- 2 degrees C, in the presence of 50 mM phosphate and 0.1 mM EDTA is (2.6 +/- 0.1) X 10(5) M-1 s-1. Using this value, 1 unit of superoxide dismutase activity (McCord, J. M., and Fridovich, I. (1969) J. Biol. Chem. 244, 6049-6055) is calculated to be 3.6 +/- 0.3 pmol of enzyme if the assay is performed in a total volume of 3.0 ml. Copper ions reduce the yield of the reaction of ferricytochrome c with CO2.(-). The reactivities of native and singly modified 4-carboxy-2,4-dinitrophenyllysine cytochromes c towards the superoxide anion radical are in the order native greater than 4-carboxy-2,4-dinitrophenyllysine 60 greater than lysine 13 greater than lysine 87 greater than lysine 27 greater than lysine 86 greater than lysine 72, indicating that electron transfer takes place at or close to the solvent accessible heme edge. The mechanism of the reaction is discussed in terms of the approach of superoxide anion radicals to the heme edge and the available molecular orbitals of both heme and free radicals.

Animals↗
PubMed1982

The asymmetric distribution of charges on the surface of horse cytochrome c. Functional implications.

The electric potential field around native horse cytochrome c and 12 singly modified 4-carboxy-2,4-dinitrophenyl- (CDNP) lysine cytochromes c is asymmetric, mainly because of the inhomogeneous distribution of negative charges. Dipole moments of 325 and 308 debye, (1.08.10(-27) and 1.03.10(-27) coulomb.meter), respectively, were calculated for horse ferri- and ferrocytochrome c. The angle between the heme plane and the dipole vector of horse ferricytochrome c is 33 degrees and increases 1 degree upon reduction to the ferrous form. Dipole moments of the CDNP-lysine cytochromes c differ from that of native cytochrome c by as much as 140 debye in magnitude and 45 degrees in direction. It is proposed that its dipole moment causes cytochrome c to orient itself in the electric fields of its redox partners, and that the CDNP-lysine cytochromes c, which have different dipole moments, do not form a productive complex. Reorientation to the correct position for electron transfer increases the activation energy and lowers the rate of reaction. This model describes quantitatively the relative activities of those CDNP-lysine cytochromes c which are modified outside of the interaction domain and it allows correction of the activities of those modified inside the domain, on the front surface of the molecule, for the change in dipole moment. The interaction domain for the reaction with cytochrome c reductase includes in decreasing order of involvement lysines 13, 72, 86, 27, and 87. That for the reaction with cytochrome c oxidase is slightly smaller, with lysines 13, 72, 86, and 27. The cytochrome c peroxidase domain is the largest of all and is defined by lysines 72, 86, 13, 87, 27,, and 73. All refined interaction domains encompass the exposed heme edge and are to a large extent overlapping, indicating that electron transfer takes place at or close to this prosthetic group and that cytochrome c must move on the outer surface of the inner mitochondrial membrane during electron transport between reductase and oxidase. For a quantitative description of the electrostatic interaction of cytochrome c with other molecules, it is essential to take into account the totality of its charge configuration.

Amino Acid Sequence↗
PubMed1982

The major B and T cell determinant on pigeon cytochrome c in B10.a mice.

The specificities of B10.A B and T cells responding to pigeon cytochrome c have been examined. Proliferating T cells recognize glutamine 100 and lysine 104 and can be stimulated in vitro by either native cytochrome c molecules or certain of their CNBr-cleavage fragments. In contrast only molecules with the native cytochrome c conformation were found to interact with B10.A antipigeon cytochrome c antibodies. Antibodies appear to recognize a determinant or determinants which overlaps with that which elicits the T cell proliferative response.

Animals↗
PubMed1981

Protein influences on porphyrin structure in cytochrome c: evidence from Raman difference spectroscopy.

To probe the details of protein heme interactions, we have developed a Raman difference spectroscopic technique, which allows reliable detection of very small, approximately equal to 0.01 cm-1, frequency differences. When this technique is applied to heme proteins, structural differences in the protein which perturb the porphyrin macrocycle may be examined by obtaining Raman difference data on the porphyrin vibrational modes which are strongly enhanced in the Raman spectrum produced with visible laser excitation. We report here Raman difference spectroscopic data on cytochromes c from 24 species. The differences in the Raman spectrum of the porphyrin between the cytochromes c of any two species are small, confirming that all of the cytochromes we have examined have the same "cytochrome fold". However, many small (0.02-2 cm-1) but systematic differences were detected which indicate structural differences among these proteins. These differences could be classified into three different groups and interpreted in terms of different types of structural variations resulting from specific differences in the amino acid sequences. First, direct interactions between near-heme residues and the porphyrin influence the electron density in the pi orbitals of the porphyrin macrocycle. Second, variation in the residue at position 92, far removed from the heme, affects the frequency of the core-size marker line at 1584 cm-1. Third, the conformation near cysteine 14 affects the shape of the Raman mode which is sensitive to the pyrrole ring substituents (approximately 1313 cm-1). From these data we conclude that there are several ways in which the protein amino acid sequence may regulate the oxidation-reduction potential and several ways in which the sequence can modify the binding site between cytochrome c and its redox partners.

Animals↗
PubMed1981

Electron paramagnetic and electron nuclear double resonance of the hydrogen peroxide compound of cytochrome c peroxidase.

We have collected electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectra from the hydrogen peroxide compound of yeast cytochrome c peroxidase, termed ES, employing EPR microwave frequencies of 9.6 and 11.6 GHz. We have measured and analyzed the temperature dependence of the spin-lattice relaxation rate (1/T1) of the paramagnetic center of ES over the temperature range 1.9 to 4 K. In addition, an upper bound to exchange coupling between the ferryl heme and EPR-visible centers of ES has been calculated and expressions for the dipolar interaction between a ferryl heme and a free radical have been derived. These results all confirm that the EPR signal of ES is not associated with an aromatic amino acid radical, and in particular not with a tryptophanyl radical. This conclusion has led us to consider an explanation of the EPR signal in terms of a nucleophilically stabilized methionyl radical.

Binding Sites↗
PubMed1981

In vitro synthesis and posttranslational uptake of cytochrome c into isolated mitochondria: role of a specific addressing signal in the apocytochrome.

Administration of the thyroid hormone 3,3,5'-triiodo-L-thyronine (T3) to rats leads to a marked increase in hepatic levels of mRNA for cytochrome c. Messenger RNA prepared from the free polysomes of T3-treated rats directed the in vitro synthesis of a polypeptide which only differed in amino acid sequence from mature cytochrome c in that it contained an NH2-terminal methionine. The in vitro product was incorporated specifically into purified rat liver mitochondria and became inaccessible to added trypsin when the mitochondria were added after translation was completed. Horse heart apocytochrome c, but not the holocytochrome, could compete with the in vitro synthesized polypeptide for its uptake into mitochondria. This suggests that the primary structural features of apocytochrome c, which serve as an addressing signal for mitochondria, are masked after the acquisition of heme and that this process occurs in the mitochondria. The addressing signal seems to be contained in a specific segment of the cytochrome polypeptide because only one fragment generated by CNBr cleavage of horse apocytochrome c, extending from residue 66 to the carboxy end of the molecule, could compete with the in vitro product for its transfer into mitochondria.

Animals↗
PubMed1981

Parallel cross-reactivity patterns of 2 sets of antigenically distinct cytochrome c peptides: possible evidence for a presentational model of Ir gene function.

B10.A mice were immunized with either the carboxyl terminal peptide fragment 81-104 of pigeon cytochrome c or its acetimidyl derivative and an immune response was seen with strong preference for the immunogen. Strain distribution studies and blocking with an anti-Ia monoclonal antibody indicated that the same immune response (Ir) gene and restriction element were utilized in both responses. The specificity of the responses were evaluated by restimulating in vitro with a set of cytochrome c fragments from various species. Even though the derivatized and native fragments were poorly cross-reactive, the same phylogenetic pattern was seen when pigeon cytochrome c fragment 81-104 primed cells were tested with the set of underivatized fragments and when acetimidyl pigeon cytochrome c fragment 81-104 primed cells were tested with the same set of derivatized fragments. Primed cells from a 2nd major histocompatibility complex congenic strain of mice, B10.A(5R), displayed equivalent discrimination between derivatized and native forms but showed a markedly different phylogenetic pattern of cross-reactivity. These data indicate that the immune system recognizes 2 sites on the nominal antigen. One site, which accounts for the common hierarchy and is under Ir gene control, contains residues Gln-100, and possibly other carboxyl terminal residues. The 2nd site, which effects the distinction between native and derivatized fragments, contains at least 1 lysine other than at the carboxyl terminal. The implications of these data for theories of T cell recognition and Ir gene function are discussed.

Animals↗
PubMed1980

Copper electron-nuclear double resonance of cytochrome c oxidase.

Electron-nuclear double resonance of copper was observed while monitoring the "intrinsic copper" electron paramagnetic resonance signal of cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) near g = 2. This unambiguously establishes the presence of the metal (Cua) in the redox center responsible for this signal. The hyperfine couplings to copper are largely istropic and the maximum value is about half that seen in type I blue copper proteins. The magnetic properties of this oxidized copper center are not consistent with those of a thiyl radical (R-S) coordinated to Cu(I), and thus favor the identification of this redox center as a Cu(II) ion in a unique environment.

Animals↗