Bioenergetics — Identify the experimental observation that does NOT support Peter Mitchell’s chemiosmotic theory of oxidative phosphorylation in mitochondria.

Introduction / Context:
Mitchell’s chemiosmotic theory proposes that electron transport chains pump protons across the inner mitochondrial membrane, creating an electrochemical gradient (proton-motive force). ATP synthase then uses this gradient to synthesize ATP. Any observation that ties ATP synthesis to the proton gradient supports the theory, whereas results that disconnect ATP from the gradient or from electron transport would argue against it.

Given Data / Assumptions:

• Mitochondria pump H+ across the inner membrane during electron transport.
• Uncouplers (e.g., dinitrophenol, DNP) collapse the proton gradient by carrying H+ across the membrane.
• ATP synthase requires a proton motive force (pmf) to function.
• Artificially imposed H+ gradients can substitute for electron transport to drive ATP synthesis.

Concept / Approach:
The theory predicts two key dependencies: (1) electron transport should establish a transmembrane H+ gradient; and (2) ATP synthesis should require that gradient. Thus, anything that dissipates the gradient should inhibit ATP synthesis, even if electron transport continues. Conversely, creating a gradient without electron transport should still yield ATP via ATP synthase.

Step-by-Step Solution:

Verification / Alternative check:
Classic reconstitution experiments (e.g., bacteriorhodopsin + ATP synthase in liposomes) prove that a proton gradient alone drives ATP formation. Moreover, uncouplers increase oxygen consumption (accelerated electron transport) while blocking ATP production, demonstrating the tight dependence on pmf.

Why Other Options Are Wrong:

• Medium acidification: consistent with H+ pumping.
• DNP blocking acidification: expected if the gradient is collapsed.
• Artificial gradient driving ATP: a hallmark prediction confirmed experimentally.
• Reconstitution with pmf yielding ATP: direct validation.

Common Pitfalls:
Confusing inhibitors of electron transport (e.g., cyanide, rotenone) with uncouplers (e.g., DNP, FCCP). Uncouplers do not primarily inhibit electron flow; they short-circuit the gradient, halting ATP synthesis.

Final Answer:
Adding dinitrophenol (an uncoupler) to isolated mitochondria inhibits electron transport but does not affect ATP synthesis.