Also known as ATP synthase, F-type ATPase, or F1F0 ATPase, Complex V is the final step in the process of mitochondrial oxidative phosphorylation. With a molecular weight of approximately 500 kD, it consists of 16 subunits, two of which—ATP6 and ATP8—are encoded by mitochondrial DNA. The enzyme is divided into two main domains: F0, which acts as a proton channel and includes membrane-bound proteins such as a, b, c, d, e, F6, A6L, and OSCP; and F1, a water-soluble domain composed of α3β3γδε subunits that carries out the catalytic function. One of its most distinctive features is its sensitivity to oligomycin, which inhibits ATP synthesis. The primary role of Complex V is to generate ATP, the energy currency of the cell. This process depends on the proton gradient created by the electron transport chain during oxygen reduction in the mitochondria. Protons flow through the F0 domain into the mitochondrial matrix, triggering conformational changes in the F1 domain and driving ATP synthesis from ADP and inorganic phosphate. Mutations or dysfunctions in this enzyme can lead to serious conditions, particularly affecting the heart and nervous system. To measure Complex V activity, ATP is hydrolyzed by F1F0 ATPase in the presence of oligomycin. This reaction is then coupled with the pyruvate kinase (PK) and lactate dehydrogenase (LDH) systems. The NADH produced in these reactions is oxidized to NAD+, causing a measurable change in absorbance at 340 nm. This optical change allows for the quantitative assessment of F1F0 ATPase activity, making it a key technique in both research and diagnostic settings.
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