Question

35. Intact mitochondrial were isolated, carefully washed with pH 7.0 buffer, and used to measure the P/O ratio for α-ketoglutarate

35. Intact mitochondrial were isolated, carefully washed with pH 7.0 buffer, and used to measure the P/O ratio for a-ketoglutarate. Included in your assay for the measurements were ADP, phosphate (Pi), NAD+, and malonate in an appropriate buffer. Assume that all the added compounds can get into the mitochondrial matrix and that all enzymes of the citric acid cycle are active. Note that malonate is a competitive inhibitor or succinate dehydrogenase. Under these conditions, what would be the P/O ratio for a-ketoglutarate? A. B. C. D. E. P/O ratio = 0.5 P/O ratio = 1.5 P/O ratio = 2.0 P/O ratio = 3.5 P/O ratio = 5.0

Answer 1

P/O ratio or the Phosphate/ Oxygen ratio is the measurement of the ATP produced from the movement of electrons in the oxidative phosphorylation pathway and simultaneous reduction of oxygen into water. To understand the calculation of P/O ratio, one needs to study the oxidative phosphorylation pathway in detail. OXPHOS involves the reduction of oxygen to water by the electrons donated from NADH and FADH. The electrons from all the NADH and FADH produced by various metabolic pathways are channelled through transmembrane complexes present in the inner mitochondrial membrane. This creates a proton motive force which drives ATP production. In this question, α-ketoglutarate is added in the buffer which enters citric acid cycle to yield succinyl CoA as the next product. Succinyl CoA gets converted to succinate which cannot be converted to fumarate further because enzyme complex succinate dehydrogenase is blocked by malonate.

There are all total 4 transmembrane complexes (Complex I, II, III and IV) and ATP synthase (which is also known as Complex V).

Complex I: It is also called NADH:ubiquinone oxidoreductase or NADH dehydrogenase which catalyzes the transfer of a hydride ion to ubiquinone from NADH. Complex I is driven by proton pump, where 4H+ ions are transported from mitochondrial matrix to the intermembrane space.

Complex II: Succinate dehydrogenase (the enzyme which is also required in the citric acid cycle) is the one and only membrane bound enzyme in the citric acid cycle pathway. This transmembrane channel is blocked by malonate in this question. Complex I, III and IV are independent of complex II. Electrons get transferred to ubiquinone from Complex I directly, without involving Complex II.

Complex III: This complex couples the transfer of electrons from ubiquinol to cytochrome c, by pumping out four protons to the intermembrane space.

Complex IV: The final complex transfers electrons directly to molecular oxygen, reducing it to water.

ATP Synthase: The proton concentration gradient that has occurred due to electron transfer is now utilized by ATP synthase which drives the synthesis of ATP as protons flow passively back into the mitochondrial matrix.

OUTER MEMBRANE INTERMEMBRANE SPACE Cyta 4HT 4Ht INNER MEMBRANE I MATRIX H2O 2H+ + ADP VNART NADH+ H+ Ата - 3H+ CHEMIOSMOTIC MODEL 18+ ONE ADDIONAL PROTON REQUIRED TO TRANSPORT ADP and Pi

Calculation: In this question, only one molecule of NADH is produced when α-ketoglutarate is converted to succinyl CoA, and 1 ATP is formed when Succinyl CoA is converted to succinate.

NADH CH3 -Coo CH₂-Coo CH2 CH2 CH₂ - cool succinyl COA CH2 synthetase C-S-COA ADP / x-ketoglutarale dehydrogenase complex t e=0 COA-SH CO2 ATP succinate COD a-ketoglutarate succinyl CoA

CH₂ - coo coo succinate dehydrogenase CH inhibited by malonate He CH2 c00 con SOCCINATE FUMARATE

Number of protons pumped out of the mitochondrial matrix for a molecule of NADH is 10. Number of protons turned back into the matrix for ATP synthesis is 4 (3 required by ATP synthase, 1 proton required to transfer ADP and Phosphate across the membrane). NADH produces a pair of electrons which is transferred to oxygen to reduce to water. This requires 10 protons to move out, and 4 must flew in to synthesize 1 molecule of ATP.

Therefore, the P/O ratio is 10/4 = 2.5+1 (ATP) = 3.5

Correct answer is Option D