### Mitochondrial Electron Transfer and Inhibition1a. **Electron Transfer Catalysts** - Illustrate and label the sequence of mitochondrial electron transfer catalysts. Begin with the oxidation of NADH and succinate, culminating in the reduction of O₂.b. **Proton Translocation Sites** - Identify the locations and stoichiometry (per 2 electrons) where protons are moved from the mitochondrial matrix to the intermembrane space.c. **Complex Inhibition** - Determine which complexes are inhibited by the following substances: - Amytal - Antimycin A - Azide (N₃⁻) - Cyanide (CN⁻) - Carbon monoxide (CO) - Rotenone d. **Effects on Respiration and ATP Synthesis** - Examine the effects of the following on respiration and ATP synthesis when introduced to a mitochondrial suspension with excess malate, ADP, and inorganic phosphate (Pi): 1. **Oligomycin** 2. **Uncouplers of Oxidative Phosphorylation**, such as: - Dinitrophenol (DNP) - Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) This exercise explores the complex interplay of electron transfer, proton translocation, and the impact of inhibitors and uncouplers on mitochondrial function. Understanding these processes is crucial to studying cellular respiration and bioenergetics.

ETC consists of four protein complexes called Complex I, II, III and IV that transport electrons from electron donors (NADH/FADH2) to electron acceptor Oxygen. During this process of electron transfer from NADH/FADH2 to oxygen, protons are pumped into the inter membrane space to generate a proton gradient that is needed by ATP synthase to synthesize ATP. Complex I carries out the oxidation of NADH. e- are transferred from NADH → FMN → Fe-S clusters → Q to reduce coenzyme Q to QH2. QH2 is lipid soluble so it diffuses within the membrane to Complex III. During this process, complex I pumps 4H+ from the matrix to IMS.NADH + 6H+(from matrix) + Q → NAD+(in matrix) + QH2(to complex III) + 4H+(in IMS) FADH2 skips Complex I and donates the electron it is carrying to Complex II.. Complex II contains succinate dehydrogenase (TCA cycle) that oxidises succinyl CoA to fumarate while reducing FAD+ to FADH2. Electron travels from FADH2 to Fe-S culture to coenzyme Q and reduces it to QH2. QH2 diffuses to Complex III. No hydrogen are pumped by this complex.Complex III catalyses the transfer of e- from QH2 to cytochrome c; meanwhile, complex III pumps 4H+ from matrix to IMS. (2 from the matrix, 2 from QH2) QH2 + 2 cytochrome c1(oxidised) + 2 H+(from matrix) → Q + 2 cytochrome C1 (reduced) + 4 H+ (IMS)Cytochrome c carries e- from complex III to complex IV.Complex IV also known as Cytochrome oxidase, catalyses the transfer of e- from cytochrome c1 to molecular oxygen and pumps 2 H+ from the matrix into the IMS.4 cytochrome c (reduced) + 5 H+(from matrix) + O2 → 4 cytochrome c (oxidised) + 1 H+(in IMS) + 2H2OMechanism of splitting Oxygen:Complex IV has two heme groups: a and a3 and two Cu centres: CuA CuB. Electrons move from cytochrome c → CuA → heme a → heme a3−CuB → O22H+ + 1/2 O2 → H2OFor every 4 e- that pass through this complex 4 H+ are pulled from the matrix to combine with 2 O2- to form H2O and uses the energy to pump 1 H+ to IMS. The image below illustrates the different complex and their function, inhibition in ETC.Oxidation of glucose, citrate etc generates electrons that are carried to the ETC via NADH and FADH2. ETC oxidises these carries to generate a proton gradient while reducing oxygen to water. The energy in proton gradient drives ATP synthesis. ATP is synthesised by phosphorylation of ADP with Pi. Since oxidation of different substrates generates electrons that establish proton gradient used for ATP synthesis, the process is called oxidative phophorylation. Effect of oligomycinMitochondrial ATP synthase is made up of two subunits: Fo and F1. Fo subunit is an integral membrane protein while F1 is a peripheral membrane protein. As per the chemiosmotic model, the proton motive force drives the ATP synthesis. ATP synthase has two domains F1 and Fo. The Fo (o denotes inhibition due to oligomycin) unit is the proton channel. By the rotational catalysis mechanism, three active sites of F1 take turns catalysing ATP synthesis from ADP and Pi. When oligomycin is added, the Fo subunit is inhibited and no protons flow through. This prevents F1's rotational catalysis and no ATP synthesis occurs. Effect of decouplersIn absence of decouples, protons in the IMS must go through the ATP synthase complex to reenter mitochondiral matrix. As they enter matrix, ATP synthase uses the energy they release to synthesise ATP. Decouples are chemical agents that allow the protons to enter the matrix without having to go through the ATP synthase pathway. As as result the proton gradient necessary for ATP synthesis does not build up and ATP synthesis halts. Note that the malate is necessary for NADH (from glycolysis) transport into the matrix. ADP and Pi are substrates for ATP synthase.A verbal description of ETC is provided in step 1 and step 2 illustrates the ETC, its stoichometery and different inhibitors. The effects of oligomycin and decouplers are described in step 3.