Mitochondrial Electron Transfer Electron transport in mitochondria.

The Big Picture (One last time) The major paths that produce NADH and FADH2. The electrons are transferred to oxygen.

Mitochondria Electron transport occurs in the mitochondria - an organelle. Enzymes are located in particular substructures of the mitochondria as listed.

Evolutionary Origin of Mitochondria Mitochondria presumably came from bacteria.

Mitochondrial Electron Carriers NADH and FADH2 (Flavoproteins) Dehydrogenases Iron-sulfur proteins Cytochromes Ubiquinone (Coenzyme QCoQ) In inner mitochondrial membrane Highly-organized complexes Important points about mitochondria.

Iron-Sulfur Proteins (Fe-S) The proteins of the complex contain functional iron-sulfur proteins that undergo oxidation and reduction.

Cytochromes Heme in Cytochrome c Cytochromes carry electrons.

Mitochondrial Cytochromes One electron carriers Fe+3 + e–  Fe2+ Eo depends on associated proteins There are cytochromes with different structures and different redox potentials.

Ubiquinone  UQ  CoQ A lipid soluble carrier moves freely in the lipid membrane.

Order of Mitochondrial e– Carriers Detection of oxidized, reduced forms Visible spectra (400-600 nm) epr spectra (Fe-S proteins) Order by Eo Kinetics of oxidation Effects of specific inhibitors Biochemical dissection of complexes How do you determine the order of functioning of the carriers?

Carrier Order by Oxidation Kinetics 1. Mitos with NADH, no O2: all reduced 2. At time zero, add O2 Order of reduction.

Carrier Order by Inhibition Specific inhibitors act at specific sites. Carriers ahead of block are reduced Carriers behind block are oxidized

Carrier Order by Dissection Take the mitochondria apart need detergent. Karp, Gerald. Cell and Molecular Biology, 2e. 1999.

Electron-Transfer Complexes: H+ Pumps A picture of the organization As electrons flow to O2, H+ flows out

Complex III A protein from complex III.

Complex IV One from complex IV.

Cu Center of Complex IV The Cu center in complex IV.

Respiration in Mitochondria 3 reactions Substrate is oxidized O2 consumed (H2O) ADP + Pi  ATP Oxidation, oxygen consumption, and ATP formed in parallel.

Oxidation and Phosphorylation Coupled processes Block electron flow (CN–, rotenone, etc)  no ATP formation Block ATP synthesis (no ADP, or inhibitor of ATP synthase-oligomycin) no electron flow Oxidation and phoshorylation are connected processes.

Uncoupling Agents and Conditions Allow electron flow with ATP synthesis Example: 2,4-dinitrophenol (DNP) Others: CCCP, ionophores, membrane disruption 2,4-Dinitrophenol is a classic uncoupling inhibitor that allows electron flow to continue but ATP is not made. Some medical doctors thought that it would be a good diet pill; burn calories and not make ATP. The patients not only lost weight but also their lives.

Efficiency of Oxidative Phosphorylation Lots of energy available.

Observed P/O Ratios But the current accepted is lower; 2.5 (2.3) and 1.5 (1.4).

Phosphorylation “Substrate-level” vs. “Respiration-linked” Two kinds of phosphorylation - substrate during glycolysis and etc linked.

Bimetallic Fe-Cu Reaction Center In cytochrome oxidase Bimetallic electrodes. Karp, Gerald. Cell and Molecular Biology, 2e. 1999.

Cytochrome Oxidase http://www.pigment.unl.edu/bioc933/ Path of electrons in cytochrome oxidase. Within each of the component parts of the chain there are several sites for redox reactions. http://www.pigment.unl.edu/bioc933/ metalloenzymes/Cyt%2OOxidase/ _COX_cycle.gif

Order of Carriers: Ascending Eo The order of the carriers.

Reduced Forms of O2 Various reduced forms of oxygen.

Enzymatic Defenses Against O Oxygen radicals can be toxic - enzymes destroy.

Vitamin E (Tocopherol) Radical scavenger Vitamin E is a radical scavenger and can prevent the accumulation of oxidized products. Aging brings oxidation and reducing agent lessen the oxidations.

The FoF1 Complex The structure of the ATP synthase, complex V.