Interaction of cytochrome c with cytochrome c oxidase studied by monoclonal antibodies and a protein modifying reagent

Abstract

C/57 black mice were immunized with beef heart cytochrome c oxidase, generating 48 hybrid cell lines that secrete antibodies against the different subunits of the enzyme. Immunoblot analysis showed reactions with 7 of the 13 subunits. Among the monoclonal antibodies produced, only those to subunit II gave significant inhibition; these inhibited the enzyme activity completely and prevented cytochrome c binding to the enzyme. Epitope mapping studies indicate that a peptide including residues 200-227 reacts with the antibody, suggesting that the C-terminus of the protein is essential for the binding of this antibody. The carboxyl modifying reagent 1-ethyl-3-[3-(trimethylammonio)propyl]carbodiimide (ETC) was chosen to investigate further the relationship between antibody and cytochrome c binding domains. ETC caused 50% inhibition of the enzyme activity with a first-order time during the first 20 min; a slower reaction over 3 h resulted in 90% inhibition. Cytochrome c binding to the oxidase was inhibited to a similar extent as cytochrome c oxidation, and protection against both effects was afforded by the presence of cytochrome c during ETC modification. Anion-exchange of FPLC of the modified forms of cytochrome oxidase revealed extensive inhomogeneity, indicating random derivatization of a number of different carboxyls even during the first-order reaction, and precluding identification of carboxyl residues related to a specific phase of the reaction. Cytochrome c and the subunit II-specific antibody protected against radioactive labeling of subunit II by ETC in the presence of [14C]glycine ethyl ester, demonstrating that the antibody and cytochrome c occupy significant and overlapping areas on the subunit II surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of YpmQ, an Accessory Protein Required for the Expression of Cytochrome c Oxidase in Bacillus subtilis

“…Western Blots with Anti-His Tag and Anti-caa 3 Antibodies-Membrane proteins of B. subtilis were extracted in Triton X-100 as described (22). Extracted proteins were separated by SDS-PAGE (32) and blotted to PVDF membrane using a mini trans-blot cell (Bio-Rad).…”

“…In cytochrome c oxidases, such as the enzyme found in the mitochondrial inner membrane of eukaryotes, reducing equivalents are delivered from the soluble protein ferrocytochrome c. The cytochrome c interaction site on the oxidase is predominantly defined by subunit II (2,3). Subunit II has two transmembrane helices that anchor a solvent-exposed domain, which provides the inner sphere ligands for the dinuclear Cu A center.…”

Characterizing monoclonal antibody structure by carbodiimide/GEE footprinting

“…The carbodiimidelabeling method with GEE tagging has previously been used to probe the structure of a number of proteins, such as the mammalian polyamine transport system and the membrane-attached antenna protein (including mapping a protein-protein interface), and to study phosphorylation-induced conformation changes of a membrane associated kinase. [44][45][46][47][48][49] For this work, we evaluated the reproducibility of protein labeling kinetics at the specific side chain residues with this reagent using a mAb. Specifically, the mAb was exposed to carbodiimide in the presence of GEE label for varying amounts of time from 0 to 10 minutes in triplicate and at various concentrations, providing a detailed quantitative characterization of side chain reactivity.…”

Identification of Essential Amino Acids within the Proposed CuA Binding Site in Subunit II of Cytochrome c Oxidase

“…Mutations , closely situated in the primary sequence between the two conserved cysteines, as a surface-exposed participant in the binding site for cytochrome c (13,28). Structural studies of cytochrome c and its physiological partner cytochrome c peroxidase show interactions between positively charged amino acids on the surface of cytochrome c and negatively charged amino acids on cytochrome c peroxidase (46).…”

“…Similarly, a monoclonal antibody to subunit II inhibits cytochrome c binding and protects regions of subunit II from reaction with the carbodiimide (28). One of the protected carboxylates, Glu-198, is rigorously conserved and is located directly between the two conserved Cys residues at 196 and 200.…”