Chapter 19 Oxidative Phosphorylation Electron transferring (flow ) through a chain of membrane bound carriers (coupled redox reactions), generation of a transmembrane proton gradient, ATP biosynthesis (ADP phosphorylation). For Biochemistry II lectures of Nov. 26 and Dec. 3, 2008 (Prof. Zengyi Chang)‏ “Anyone who is not confused about oxidative phosphorylation just doesn’t understand the situation” Efraim Racker, 1970s

*all the electrons are transferred to O2; *ATP is made using a proton gradient. Main events: The three stages of biological oxidation Coupling of energy-releasing & energy-requiring reactions. Energy-requiring Energy-releasing

History of understanding oxidative phosphoryation 1900s: vital role of phosphate in fermentation revealed S: role of iron in intracellular respiration realized. 1920s: Cytochromes identified and the concept of the respiratory chain (electron transport chain) formulated. 1930s: pyruvate (product of glycolysis) known to be completely oxidized to CO 2 via the citric acid cycle (needing O 2 ). 1930s: NAD + and FAD were found to be e - carriers between metabolites and the respiratory chain. 1930s: role of ATP and general importance of phosphorylation in bioenergetics realized (Lipmann, 1939, “oxidative phosphorylation” introduced)‏

A historical perspective Understanding the detail molecular process 1940s: link between sugar oxidation and ATP synthesis established; Role of NADH linking metabolic pathway and ATP synthesis proved. 1950s: isolated mitochondria found to effect the obligatory coupling of the phosphorylation of ADP and the e - transfer from NADH to O s-1950s: “High energy intermediate” leading to ATP synthesis (chemical coupling hypothesis) was searched (but failed to be found.) 1960s: the chemiosmotic hypothesis proposed for linking the e - transfer and ADP phosphorylation (role of the membrane and the across-membrane proton gradient postulated, a new paradigm!).‏ 1970s: the binding change model was proposed to explain how the proton gradient will be used to drives ADP phosphorylation. 1990s: structure of ATP synthase determined, supporting the binding change model.‏

Electrons of NADH and FADH2 are transferred to O 2 via many intermediate electron carriers making up the respiratory chain.

History of understanding Photophosphoryation 1790s: CO 2 and H 2 O are taken up by green plants, while O 2 is released, all under the influence of light. 1930s: photosynthesis occur via light-dependent redox reaction; isolated chloroplasts made O 2 but did not fix CO 2 ; O 2 is derived from H 2 O. 1950s: The path of carbon fixation revealed. 1960s: Existence of two photosystems elucidated.

NADH enters at NADH-CoQ oxidoreductase Consisting ~34-46 polypeptide chains The coupling mechanism between e - transferring and H + pumping is still unknown! ~880 KDa （ the largest among four ） NADH: 2e FAD:1e FMN 1e or 2 e

At least eight different types of iron-sulfur centers (first revealed by Helmut Beinert) act in the respiratory chain: (in complex I, II and III) iron atoms cycle between Fe 2+ (reduced) and Fe 3+ (oxidized). 2Fe-2S 4Fe-4S

Ubiquinone (or coenzyme Q) is the only e - carrier that is not bound to a protein and is able to diffuse freely in the lipid bilayer. (or dihydroubiquinone)‏ The isoprenoid tail Q10

FADH 2 of flavoproteins also transfer their electrons to ubiquinone (Q), but with no H + pumped. Succinate dehydrogenase

Structure of mitochondrial complex II (porcine) was determined Sun F et al. and Rao, Z, 2005, Cell, 121: Proposed electron path Succinate dehydrogenase 孙飞

The crystal structure of the cytochrome bc 1 complex (Complex III) has been determined. Structure of the three core subunits out of the 11 subunits. Spatial relationship of the cofactors deduced from the resolved structure of complex III. Science, 1997, 277:60-66.

Three types of heme groups are found in the cytochromes. Bound to cytochromes tightly and covalently Also found in Hemoglobin & myoglobin Bound to cytochromes tightly but noncovalently Bound to cytochromes tightly but noncovalently The iron interconverts between its reduced (Fe2+) and oxidized (Fe3+) froms, thus performing oxidation and reduction reactions

Reduced cytochromes has three absorption bands in the visible wavelengths The reduced (Fe 2+ )‏ state of cytochromes a, b, and c has the longest wavelength band near 600, 560, and 550 nm respectively. cba Cytochromes are classified on the basis of position of their lowest energy absorption band in the reduced state.

The e - transferring & H + pumping in Complex III occur via the Q cycle For each 2 e - transferred, 4H + are translocated.

the Q cycle From:

Cytochrome c oxidase (Complex IV ), contains 3 Cu and 2 heme A groups as electron carriers. Heme a and heme a 3 has identical structures but different reduction potential. The three critical subunits (out of 13) of complex IV. a a3a3 Cu A -Cu A Cu B O 2 acts as the final electron acceptor here. Science, 1996,272:

Four electrons are transferred from 4 Cyt c to 1 O 2 to make 2H 2 O in Complex IV, with 4H + taken from the matrix to make 2H 2 O & 4H + pumped out.

~ 10 protons per NADH and ~6 protons per FADH 2 oxidized are pumped across the inner membrane of mitochondria (or plasma membrane of bacteria)‏ The highly mobile Q and Cyt c molecules shuttle electrons from one large multiprotein complex to another. A transmembrane H + gradient is thus generated. The electron motive force is converted to an proton motive force.