Ribose 5-Phosphate Glycation Reduces Cytochrome c Respiratory Activity and Membrane Affinity

Spontaneous glycation of bovine heart cytochrome c (cyt c) by the sugar ribose 5-phosphate (RSP) weakens the ability of the heme protein to transfer electrons in the respiratory pathway and to bind to membranes. Trypsin fragmentation studies suggest the preferential sites of glycation include Lys72 and Lys87/88 of a cationic patch involved in the association of the protein with its respiratory chain partners and with cardiolipin-containing membranes. Reaction of bovine cyt c with RSP (50 mM) for 8 h modified the protein in a manner that weakened its ability to transfer electrons to cytochrome oxidase by 60%. An 18 h treatment with RSP decreased bovine cyt c's binding affinity with cardiolipin-containing liposomes by an estimated 8-fold. A similar weaker binding of glycated cyt c was observed with mitoplasts. The reversal of the effects of RSP on membrane binding by ATP further supports an A-site modification. A significant decrease in the rate of spin state change for ferro-cyt c, thought to be due to cardiolipin insertion disrupting the coordination of Met to heme, was found for the RSP-treated cyt c. This change occurred to a greater extent than what can be explained by the permanent attachment of the protein to the liposome. Turbidity changes resulting from the multilamellar liposome fusion that is readily promoted by cyt c binding were not seen for the RSP-glycated cyt c samples. Collectively, these results demonstrate the negative impact that LW glycation can have on critical electron transfer and membrane association functions of cyt c.