Abstract
The reagent 1-ethyl-3-(3-[14C]trimethylaminopropyl)carbodiimide (ETC) was used to identify specific carboxyl groups on the cytochrome bc1 complex (ubiquinol-cytochrome c reductase, EC 1.10.2.2) involved in binding cytochrome c. Treatment of the cytochrome bc1 complex with 2 mM ETC led to inhibition of the electron transfer activity with cytochrome c. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that both the cytochrome c1 heme peptide and the Mr = 9175 "hinge" peptide were radiolabeled by ETC. In addition, a new band appeared at a position consistent with a 1:1 cross-linked cytochrome c1-hinge peptide species. Treatment of a 1:1 cytochrome bc1-cytochrome c complex with ETC led to the same inhibition of electron transfer activity observed with the uncomplexed cytochrome bc1, but to decreased radiolabeling of the cytochrome c1 heme peptide. Two new cross-linked species corresponding to cytochrome c-hinge peptide and cytochrome c-cytochrome c1 were formed in place of the cytochrome c1-hinge peptide species. In order to identify the specific carboxyl groups labeled by ETC, a purified cytochrome c1 preparation containing both the heme peptide and the hinge peptide was dimethylated at all the lysines to prevent internal cross-linking. The methylated cytochrome c1 preparation was treated with ETC and digested with trypsin and chymotrypsin, and the resulting peptides were separated by high pressure liquid chromatography. ETC was found to label the cytochrome c1 peptides 63-81, 121-128, and 153-179 and the hinge peptides 1-17 and 48-65. All of these peptides are highly acidic and contain one or more regions of adjacent carboxyl groups. The only peptide consistently protected from labeling by cytochrome c binding was 63-81, demonstrating that the carboxyl groups at residues 66, 67, 76, and 77 are involved in binding cytochrome c. These residues are relatively close to the heme-binding cysteine residues 37 and 40 and indicate a possible site for electron transfer from cytochrome c1 to cytochrome c.
Highlights
From the $Departmentof Chemistry, University of Arkansas, Fayetteuilk Arkansas 72701 and the qDepartment of Biochemistry, Agricultural Experiment Station, Oklahoma State University, Stillwater, Oklahoma 74078
Treatment of a 1:l cytochrome b c l cytochrome c complex with ETC led to the same inhibition of electron transfer activity observed with the uncomplexed cytochrome bcl, but to decreased radiolabeling of the cytochrome c1 heme peptide
This same group of lysine residues is involved in the interaction with cytochrome c peroxidase (Kang et al, 1978;Smith and Millett, 1980),cytochrome b5 (Stonehuerner et al, 1979),sulfite oxidase (Webb et al, 1980), and adrenodoxin (Geren and Millett, 1981).X-ray crystallographic studies have identified a ring of negatively charged carboxylates surrounding the heme crevice of both cytochrome c peroxidase and cytochrome bg that is complementary to thering of positively charged lysine residues on cytochrome c (Poulos and Kraut, 1980; Salemme, 1976)
Summary
Materiuki-Horse heart cytochrome c (Type VI), l-ethyl-3-(3-dimethylaminopropyl)carbodiimide,octyl glucopyranoside,trypsin, and chymotrypsin were obtained from Sigma. The cytochrome bcl complex was purified according to the procedure ofYu and Yu (1980), while cytochrome CI was purified according to Yu et al (1983). Into small pieces, and eluted with 0.1 M sodium phosphate containing 0.1%octyl glucoside for 7 hat 25 “C.The extract was dialyzedagainst 1mM sodium phosphate, pH 7.0, and concentrated under nitrogen to 37 PM cytochrome c1 and 10 mM sodium phosphate. The samples were digested and eluted on the RP-300 column as described above. Methylated cytochrome c or c1 was prepared by treating 5 mg/ml of the protein in 0.2 M sodium phosphate, pH 7.0, with 60 mM dimeth-
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