Covalent cross-linking of the active sites of vesicle-bound cytochrome b5 and NADH-cytochrome b5 reductase.
A water-soluble carbodiimide has been used to promote the formation of amide bonds between carboxyl residues on cytochrome b5 and lysyl residues on cytochrome b5 reductase. The visible and UV absorption spectrum of the purified cross-linked complex was identical with the sum of the spectra of the individual enzymes, and the average apparent molecular weight of the complex, determined by sodium dodecyl sulfate-gel electrophoresis, was within 12% of the sum of the apparent molecular weights of the two monomeric enzymes, indicating that the cross-linked derivative was a dimer containing one molecule each of cytochrome b5 and cytochrome b5 reductase. When reconstituted into phospholipid vesicles, the amphipathic derivative showed substantially reduced Vmax values with the soluble electron acceptors potassium ferricyanide, cytochrome b5 heme peptide and cytochrome c, and with the membrane-bound acceptors amphipathic cytochrome b5 and stearyl-CoA desaturase. The soluble catalytic fragment of the derivative, produced by limited digestion with subtilisin Carlsberg, showed similar decreases in Vmax values with the above soluble acceptors. In contrast, intradimer electron transfer in the soluble fragment, measured by stopped flow spectrophotometry at 2 degrees C was very efficient. Ninety per cent of the cytochrome b5 in the derivative was reduced with a first order rate constant of 51 s-1 upon the addition of NADH; the transfer of electrons from NADH to the reductase FAD prosthetic group, which is known to be the rate-limiting step in the reductase reaction mechanism, proceeded with an apparent rate constant of 57 s-1 under these conditions. These kinetic data show that the enzymes in the complex are cross-linked together at the surfaces involved in protein-protein contacts during electron transfer in an orientation similar to that assumed during electron transfer between the free proteins.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003579:Cytochrome Reductases / Q000378:metabolism*; D003573:Cytochrome b Group / Q000378:metabolism*; D042966:Cytochrome-B(5) Reductase; D015786:Cytochromes b5; D004134:Dimyristoylphosphatidylcholine; D005022:Ethyldimethylaminopropyl Carbodiimide; D007700:Kinetics; D008081:Liposomes; D008099:Liver / Q000201:enzymology*; D008297:Male; D010084:Oxidation-Reduction; D013053:Spectrophotometry
D003432:Cross-Linking Reagents; D003573:Cytochrome b Group; D008081:Liposomes; D015786:Cytochromes b5; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D005022:Ethyldimethylaminopropyl Carbodiimide; D004134:Dimyristoylphosphatidylcholine