The use of a water-soluble carbodiimide to cross-link cytochrome c to plastocyanin

Geren, Lois; Stonehuerner, Jacqueline; Davis, Danny J.; Millett, Francis

doi:10.1016/0005-2728(83)90025-7

Cited by 38 publications

(30 citation statements)

References 31 publications

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“…The electrostatic complex was made by mixing equimolar amounts of the two proteins in the same buffer. The covalent complex was made by a published method (Geren et al, 1983). The reaction mixture was 80 µ in each protein and 1 mM in EDC; the solvent was 5 mM MOPS buffer at pH 6.5; and the incubation lasted from 16 to 29 h at room temperature.…”

Section: Methodsmentioning

confidence: 99%

“…The latter residues occur mainly in the segment 42-45 but also at the positions 59-61 and 68 (Burkey & Gross, 1981, 1982. The close structural similarity between the electrostatic and covalent complexes cyt/pc has been claimed (Geren et al, 1983; King et al, 1985), but this claim can be examined only when the cross-linked residues in the latter complex are identified.…”

Section: Methodsmentioning

confidence: 99%

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Oxidoreduction reactions involving the electrostatic and the covalent complex of cytochrome c and plastocyanin: importance of the protein rearrangement for the intracomplex electron-transfer reaction

Peerey¹,

Kostić²

1989

Biochemistry

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Horse heart cytochrome c and French bean plastocyanin are cross-linked one-to-one by a carbodiimide [Geren, L. M., Stonehuerner, J., Davis, D. J., & Millett, F. (1983) Biochim. Biophys. Acta 724, 62] in the same general orientation in which they associate electrostatically [King, G. C., Binstead, R. A., & Wright, P. E. (1985) Biochim. Biophys. Acta 806, 262]. The reduction potentials of the Fe and Cu atoms in the covalent diprotein complex are respectively 245 and 385 mV vs NHE; the EPR spectra of the two metals are not perturbed by cross-linking. Four isomers of the covalent diprotein complex, which probably differ slightly from one another in the manner of cross-linking, are separated efficiently by cation-exchange chromatography. Stopped-flow spectrophotometric experiments with the covalent diprotein complex show that the presence of plastocyanin somewhat inhibits oxidation of ferrocytochrome c by [Fe(CN)6]3- and somewhat promotes oxidation of this protein by [Fe(C5H5)2]+. These changes in reactivity are explained in terms of electrostatic and steric effects. Pulse-radiolysis experiments with the electrostatic diprotein complex yield association constants of greater than or equal to 5 X 10(6) and 1 X 10(5) M-1 at ionic strengths of 1 and 40 mM, respectively, and the rate constant of 1.05 X 10(3) s-1, regardless of the ionic strength, for the intracomplex electron-transfer reaction. Analogous pulse-radiolysis experiments with each of the four isomers of the covalent diprotein complex, at ionic strengths of both 2 and 200 mM, show an absence of the intracomplex electron-transfer reaction. A rearrangement of the proteins for this reaction seems to be possible (or unnecessary) in the electrostatic complex but impossible in the covalent complex.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Oxidoreduction reactions involving the electrostatic and the covalent complex of cytochrome c and plastocyanin: importance of the protein rearrangement for the intracomplex electron-transfer reaction

Peerey¹,

Kostić²

1989

Biochemistry

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“…The latter residues occur mainly in the segment 42-45but also at the positions 59-61 and 68 (109,110). The close structural similarity between the electrostatic and covalent complexes of cyt/pc has been claimed (2,3), but this claim can be examined only when the cross-…”

Section: Properties Of the Cyt/pc Complexes 1 Electrostatic And Covalentbindingmentioning

confidence: 99%

“…Subsequent research focused on oxidoreduction reactions involving the electrostatic and the covalent complex of horse heart cytochrome c and Phaseolus vu^aris plastocyanin, and exploring the importance of protein rearrangement for the intracomplex electrontransfer reaction. Cytochrome c and plastocyanin are cross-linked one-to-one by a carbodiimide (2), in the same general orientation in which they associate electrostatically (3). The reduction potentials of the Fe and Cu atoms in the covalent diprotein complex are respectively 245 and 385 mV versus NEE; the EPR spectra of the two metals are not perturbed by cross-linking.…”

mentioning

confidence: 99%

Cross-linked metalloproteins: Novel systems for the study of intraprotein electron-transfer reactions

Peerey¹

1990

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The most advanced technology has been used to photo graph and reproduce this manuscript from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. F. Intracomplex Electron-Transfer Reactions 1. Overview of the reactions 2. Electrostatic complex: pulse radiolysis experiments 3. Electrostatic complex; flash photolysis experiments 4. Covalent complex: pulse radiolysis experiments 5. Covalentcomplex: flash photolysis experiments G. Reactivity of the Covalent cyt^c Complexes n. Conclusions V. NOVEL COMPLEXES OF CYTOCHROME C

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“…Second, as the carbodiimide is not incorporated into the adduct, it is essentially a 'zero length' cross-linker, only capable of linking groups in close proximity to one another. The approach has been used to covalently link a number of interacting proteins where electrostatic interactions are important (Davis and Hough 1983, Geren et al 1983, Geren et al 1984, Hackett and Strittmatter 1984, Waldmeyer et al 1982). …”

Section: Introductionmentioning

confidence: 99%

Characterization of a covalently linked complex involving ferredoxin and ferredoxin: NADP reductase

Colvert

Davis

1988

Photosynth Res

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Ferredoxin and the flavoprotein, ferredoxin: NADP reductase, have been covalently linked by incubation in the presence of a water soluble carbodiimide. The cross-linking reaction yields an adduct having a 1:1 stoichiometry. The adduct has depressed levels of diaphorase and NADPH oxidase activity and is inactive in reduction of cytochrome c using NADPH as an electron donor. Thus, although similar to an adduct described by Zanetti and coworkers [J Biol Chem 259: 6153-6157 (1984)] in its stoichiometry, the adduct described herein has significantly different enzymatic properties. It is suggested that this may be a reflection of differences in the interaction between the two proteins resulting from differences in experimental conditions in which the two adducts were prepared.

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The use of a water-soluble carbodiimide to cross-link cytochrome c to plastocyanin

Cited by 38 publications

References 31 publications

Oxidoreduction reactions involving the electrostatic and the covalent complex of cytochrome c and plastocyanin: importance of the protein rearrangement for the intracomplex electron-transfer reaction

Oxidoreduction reactions involving the electrostatic and the covalent complex of cytochrome c and plastocyanin: importance of the protein rearrangement for the intracomplex electron-transfer reaction

Cross-linked metalloproteins: Novel systems for the study of intraprotein electron-transfer reactions

Characterization of a covalently linked complex involving ferredoxin and ferredoxin: NADP reductase

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