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

Abstract

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.

The cellular location and specificity of bacterial cytochrome c peroxidases

“…The cytochrome c peroxidase of S. cerevisiae was isolated as described by Pettigrew & Seilman (1982); that from Ps. aeruginosa was obtained by using the procedure of Foote et al…”

Protein surface charge effect on 3D domain swapping in cells for c-type cytochromes

“…C-type cytochromes (cyts) are heme proteins that act as electron carriers, functioning in a variety of cellular processes [1,2]. Many c-type cyts can form oligomers by 3D domain swapping (herein, domain swapping) [3][4][5][6][7][8][9][10][11].…”

Kinetics of reduction by free flavin semiquinones of the components of the cytochrome c-cytochrome c peroxidase complex and intracomplex electron transfer

“…In some cases, covalent cross-linking was used. This latter methodology has been applied to two electron transfer complexes, horse cytochrome c-Azotobacter vinelandii flavodoxin (Dickerson et al, 1985) and horse cytochrome c-yeast cytochrome c peroxidase (Pettigrew & Seilman, 1982; Waldmeyer et al, 1982;Waldmeyer & Bosshard, 1985;Bechtold & Bosshard, 1985). In the latter complex, a 16-fold decrease 1 Abbreviations: cytochrome c(III) and c(II), ferric and ferro cytochrome c, respectively; CcP(III) and CcP(II), ferric and ferro cytochrome c peroxidase, respectively; CcP(IV,R,+), peroxidase species oxidized by H202 yielding an Fe(IV) and an oxidized amino acid, R"+ (i.e., compound I); CcP(IV), peroxidase species oxidized to the Fe(IV) state without R group oxidation; EDTA, ethylenediaminetetraacetic acid; LFH", RFH", FMNH', and 5-DRFH', neutral semiquinone species of lumiflavin, riboflavin, flavin mononucleotide, and 5-deazariboflavin, respectively; NADP+, oxidized nicotinamide adenine dinucleotide phosphate; £m,7, midpoint reduction potential measured at pH 7. in the rate constant for ascorbate reduction of complexed cytochrome c was observed, as well as a 95% decrease in peroxidase activity toward exogenous cytochrome c(II).…”