Inhibition of the Links between Electron Transfer and Proton Translocation in Mitochondria

Abstract

The mechanism by which proton extrusion is linked to electron transfer in mitochondria was investigated by means of the primary amine-specific reagent fluorescamine, and of compounds obtained from the reaction of fluorescamine with simple amines (e.g. benzylamine) and with the mycosamine-containing antibiotic amphotericin B. The effect of these 'modifiers' (i.e. fluorescamine and the acyclic amine-fluorescamine compounds) on the activities of sites I1 and I11 of the electron transfer chain were assayed separately using specific inhibitors to block the action associated with the other site. Both types of modifiers inhibited the proton extrusion across the membrane to a significantly greater extent than the electron transfer process in both sites I1 and 111. I n contrast, the lactone derivative (or cyclic form) of the amine-fluorescamine compounds had no significant inhibitory effect on the proton extrusion and its associated electron transfer. These results are consistent with the hypothesis that the link between proton extrusion and electron transfer in mitochondria is indirect in nature. The results show that: (a) the links involved in sites I1 and I11 are identical or very similar in nature; (b) a covalent modification of primary amino groups in the inner membrane is not essential for the expression of these differential inhibitory effects ; (c) specific structural features in the amine-fluorescamine compounds, and in the mitochondria-fluorescamine derivatives, are crucial for the expression of the inhibitory effects. Our results contradict the 'redox loop' model of Mitchell, and are compatible with the proton pump concept for the linked proton translocation in oxidative phosphorylation.

Characterization of the Effects of Divalent Cations on the Coupled Activities of the H+-ATPase in Tonoplast Vesicles

“…However, using a detailed kinetic model for the proton movement, we found that certain treatments did not change the H+ leakage of the membrane but yielded differential effects on the coupled activities in highly purified tonoplast membrane vesicles (27,28). This result suggested that the coupling between proton pumping and ATP hydrolysis may be indirect, as proposed earlier (25).…”

Differential Inhibition of Tonoplast H+-ATPase Activities by Fluorescamine and Its Derivatives

“…A similar preferential inhibition of proton pumping was also observed in the presence of acyclic (compound II) but not cyclic derivatives (compound III) (28). Thus, the presence of H-bond forming groups in the membrane was essential for inhibition.…”

“…The direct coupling mechanism implies that at least one molecular event must be common to the pathways leading to ATP hydrolysis and proton pumping (14). Coupling could also be accomplished through an indirect linkage, such as a membrane Bohr effect advocated by some researchers (10,28). In our previous work with corn root tonoplast vesicles, we demonstrated that proton pumping exhibited a greater sensitivity to nitrate inhibition (29), temperature (27), mercury (27), and divalent cations (30), as compared to ATP hydrolysis.…”

“…The reaction products (compounds II) are highly fluorescent and contain hydroxyl, carboxyl, and carbonyl groups which are excellent H-bonding functionalities. The acyclic fluorescent products slowly recyclize to form compounds III which have lower H-bonding capability (20,28).…”

“…In our previous work with corn root tonoplast vesicles, we demonstrated that proton pumping exhibited a greater sensitivity to nitrate inhibition (29), temperature (27), mercury (27), and divalent cations (30), as compared to ATP hydrolysis. To further explore the nature of the coupling between these two processes, we used fluorescamine modification employed in previous studies of mitochondria and purple membrane systems (13,28,31).…”