Topic 11: Chemiosmosis

Flavoproteins FMN & FAD

protein bound hydrogen carriers spectral shift @ 340, 370, & 450 nm

what are the 3 enzyme complexes & the two mobile carriers

1) NADH dehydrogenase complex (complex 1) 2) cytochrome C reductase complex (complex 3) 3) cytochrome c oxidase complex (complex 4) 2 mobile carriers = ubiquinone and cytochrome C

evidence of chemiosmotic coupling obtained by

1) fractionation & reconstitution 2) pH gradient 3) bacteriorhodopsin experiment

how are ETC carriers aligned?

--> carriers are aligned linearly... via increasing Redox Potential... table from more electronegative [ - ] toward more electropositive to [ + ] and therefore by their energy differentials:

ATP Rotary motor steps (3)

1. H+ movement changes binding affinity of the synthase's F1's β's active site, thus when ADP & P bind to β'-active site, they readily condense into ATP (removed from aqueous solution Keq = 1 & ΔG close to zero, thus ATP readily forms) 2. the β-subunits change conformation* through 3 successive shapes (O-L-T) O - open - site has low affinity to bind ATP - thus releases it [see E] L - loose - ADP & P loosely bound to site [1 & 2] T - tight - ADP & P tightly bound favoring condensation without water 3. conformational changes result in rotation of subunits relative to central stalk (γ); α & β subunits of F1 form hexagonal ring that rotates around central axis. γ stalk extends from F0 & interacts with 3 β's differently as it rotates thru 360 degrees

mallery's confusing ass description of what happens

12 C-proteins reside in lipid bilayer (C-ring) C-ring is attached to γ stalk of F1 subunitH+ diffuse through Fo half-channel rotating the 12C's of the Fo ring each C protein has a half-channel space with a neg charged aspartate C's bind H+ on pms side & via shape changes each C-rotates 30o CCW next C in ring picks up H+ - thus 12 C's rotates ring cycles thru 360o release of H+ into matrix happens at end of cycle Karp 5.29 4 H+ moves ring 120o (γ stalk) shifts 120o --> β's change 4 H+ result in one ATP being made rotation of C-ring drives γ stalk through 360o & ► 3 conformations of F1 (L-T-O) to make ATP

F0 composition

3 polypeptides in ratio of 1a, 2b, and 12c's C-ring binds H+ and conformationally rotates.

F1 composition

5 polypeptides (nuclear DNA coded): 3α , 3β , 1γ , 1δ, & 1ε arranged like sections of grapefruit. 3 catalytic sites for ATP synthesis - 1 on each β subunit

Binding Charge Mechanism of ATP Synthesis

= a rotary motor -discovered via Paul Boyer 1979 Nobel

uncouplers

Compounds called uncouplers were found to collapse the pH gradient by shuttling protons back across the membrane through the compounds. One such uncoupler, dinitophenol is shown below. In the presence of the uncoupler electron transport continues, but no ATP synthesis occurs.

cytochromes

Cytochromes (a, a3, b562, b566, c1, c) "colored proteins" with bound Fe atoms [ ferric+3 ox vs. ferrous+2 red] via iron porphyrin (heme) bound protein carriers

ETC

ETC is a series of electron CARRIER MOLECULES that that transfer e-'s from a more negative redox potential to a more positive redox potential, while "driving"* protons out of the mitoplasm into perimitochondrial space. An important feature to remember is that the processes of electron transport and ATP synthesis are distinct from each other and occur in different parts of the mitochondrial membranes; but they are coupled processes.

how ATP synthase works diagram

H+ enter flow down their concentration gradient into the matrix through a one way entry open to the intermembrane space and bind to rotor subunits; only protonated subunits can rotate into the membrane away from the static channel assembly; once a full circle is completed and have returned to the static subunits, an exit channel allows them to leave into the matrix; this process causes the rotor to rotate; this rotation causes conformational changes in the F1 domain, allowing for ADP and Pi to be brought close together and the formation of ATP

history of determining structure of ATP synthase

Humbeto Fernandez (60's) sees lollipops on inner mito membranes* Efraim Racker (1966) isolates lollipop - Coupling Factor 1 - F1

homoplasmic vs heteroplastic mtDNA

Mitochondrial genomes may not be uniform across cells of the body, but vary between different tissue types. It is assumed that *from the beginning of life individuals are HOMOPLASMIC, meaning that within an individual, all the cells mitochondrial DNA (mtDNA) is the same (derived from egg cell cytoplasm).* However,recent *data suggests that each individual may be a mosaic of multiple cell [mt]DNA types, in different tissues*. Using high throughput sequencing technology, molecular geneticist Nickolas Papadopoulos of the Ludwig Center for Cancer Genetic and Therapeutics and the Johns Hopkins Kimmel Cancer Center in Baltimore and his colleagues analyzed the mitochondrial genomes of a variety of tissues in 2 different people and the lining of the colon & 10 other tissues. In every individual, the *researchers found at least 1 allele that differed between tissues*, and one individual had as many as 14 *HETEROPLASMES (varying mtDNA genomes*). Once established, these findings may also affect more practical applications in forensics science, since the mtDNA in one tissue might vary from another tissue, caution must be used when comparing a hair sample, for example, to blood. It's unclear why mtDNA is so variable. *One reason may be that mitochondria have a higher mutation rate than nuclear DNA or that the mitochondria have less effective DNA repair mechanisms*. These findings are likely to spur future studies to further characterize the diversity in mitochondrial genomes and determine the mechanism underlying the variation.

comparison of redox potentials of NAD+ (beginning of ETC) to reduction of O2 (end of ETC)

NAD+ reduction is -0.32 O2 to H20 is +0.82 more positive = more easily reduced

Proton motive force

NADH 2e- > complex 1 (NADH dehydrogenase complex) > CoQ > CoQH2 (draws 2H+ from matrix) > energy released from transported electrons is used by complex 1 to *pump 4 H+ across the inner membrane* > reduced CoQ donates 2 e- to complex 3 > the two electrons are transported through the carrier to two molecules of cytochrome c (this is coupled with the release of *four more H+ molecules into inter membrane space* from matrix) > complex 4 (cytochrome c oxidase complex) transfers electrons to ultimate e- acceptor, O2, to generate H20 and *2 more H+ are pumped through the membrane* =10H+ in inter membrane space per pair of electrons from NADH All this proton pumping in the inter membrane space results in a proton concentration gradient across the inner MM and an electric potential (negative inside positive in inter membrane space) = proton motive force

# of mitochondrial proteins made in mitochondria vs in cytoplasm; mitochondrial DNA also codes for

Only 13 out of some 1,100 mito proteins are coded in the mitochondria...the rest are coded for by nucleus & made in cytoplasm. Mitochondrial DNA also codes for some tRNA & rRNA.

Mitochondrial aerobic cell respiration driven by e- transport diagram

Oxidation-reduction (Redox) reactions involve the transfer of electrons from one substance to another. Redox reactions must occur together (in couples). One compound must lose electrons and the other gain electrons. The substance which loses electrons is called the reducing agent while the substance which gains electrons is called the oxidizing agent. The reduction potential is the measure of the ability of one compound to reduce another. For example, in the following reaction O2 is the oxidizing agent (and is reduced) and NADH is the reducing agent (and is oxidized). 1/2 O2 + NADH + H+ >>>>>> H2O + NAD+

Pyridine nucleotides NAD+

Pyridine nucleotides NAD+ ecb-14.10* & ecb 3.34b enzyme bound hydrogen carriers accept 2e's and/or protons show spectral shifts @ 340nm NADH vs. NAD

Some of the evidence supporting Mitchell's chemiosmotic hypothesis is as follows.

Some of the evidence supporting Mitchell's chemiosmotic hypothesis is as follows. 1. Electron transport generates a proton gradient. The pH measured on the outside is lower than that measured inside the mitochondria. 2. Only a proton gradient is needed to synthesize ATP. Electron transport is not required as long as there is another mechanism for generating a pH gradient. 3. A reconstitution experiment carried out by Racker & Stoeckenius (J Biol Chem 1974 Jan 25;249(2):662-3, Reconstitution of purple membrane vesicles catalyzing light-driven proton uptake and adenosine triphosphate formation) showed that the generation of a proton gradient can result in ATP synthesis in a totally artificial system. In their experiment, a mitochondrial ATPase complex from beef heart was inserted into an artificial lipid bilayer. Also inserted in this bilayer was a membrane fragment containing the protein, bacteriorhodopsin* [pic], from the purple bacteria Halobacterium, so called because the bacteriorhodopsin gives the membrane a purple color. Bacteriorhodopsin is a light-driven proton pump. Therefore, shining light on this artificial "purple membrane" formed a proton gradient, which was used by the beef heart mitochondrial ATPase to synthesize ATP.

Can uncoupling of electron transport and ATP synthesis ever be useful to an organism? -what kind of fat does this -byproduct -themogenin

The answer is probably "Yes." Such uncoupling can generate an energetically wasteful byproduct, heat. This occurs normally in many in hibernating animals, in newborn humans, and in mammals adapted to the cold. It occurs in a specialized tissue known as brown adipose tissue. An uncoupling protein called thermogenin can accomplish this uncoupling and thus allow heat to be generated.

chemisosmotic hypothesis -what is it -proposed by who

The chemiosmotic hypothesis was proposed by Peter Mitchell. This hypothesis stated that a proton-motive force was responsible for driving the synthesis of ATP. In this hypothesis, protons would be pumped across the inner mitochondrial membrane as electrons went through the electron transfer chain. This would result in a proton gradient with an lower pH in the intermembrane space and a elevated pH in the matrix of the mitochondria. An intact inner mitochondrial membrane, impermeable to protons, is a requirement of such a model. The proton gradient and membrane potential are the proton-motive force that is used to drive ATP synthesis. In effect, the pH gradient acts as a "battery" which stores energy to produce ATP.

ETC symmetry

The electron transfer chains and the ATPases are asymmetrically oriented in the inner mitochondrial membrane. An asymmetric orientation is a requirement to establish a pH gradient. A random arrangement would not result in a net gradient of protons and therefore, no proton-motive force for the synthesis of ATP.

antibiotics/ poisons and where they may block the ETC

The following diagram illustrates the linear sequence of the 4 enzyme complexes in the inner mitochondrial membrane and shows the flow of electrons within this respiratory chain and their blockage by specific poisons and/or antibiotics.

prosthetic groups include

The prosthetic groups are the units involved in the actual transfer of electrons within each complex and from one complex to another and are classified as 1) iron sulfur proteins (Fe-S), 2) hemes, 3) copper ions, and 4) flavins All of them serve to carry electrons but each enzyme complex is associated with specific prosthetic groups.

enzyme complexes are also known as

also known as respiratory assemblies. Respiratory assemblies are enzyme complexes of acceptor proteins, coenzymes, and metal ions, are located in the inner mitochondrial membrane. The respiratory assemblies are made up of 3 enzyme complexes, which are the sites of the proton pumps.

ATP synthase F0 F1

condenses ADP + Pi ---> ATP has a hydrophilic channel (F0) for H+ flow& makes 100 ATP per 300 H+ per sec F1 - 'matrix' soluble piece: 9 proteins F0 - membrane bound piece stalk: 15 proteins

pH difference and membrane potential difference (approx)

about 0.7-1.0 pH units difference delta charge is a bout 140 mV

prosthetic groups

all of these enzymes are large, multisubunit complexes containing several prosthetic groups. The prosthetic groups are the molecules that actually carry the electrons within the complex.

delta G equation relating delta G to standard cell potential

delta G0 = -nFE0

MM inner membrane-composition (%) -contains what proteins?

inner membrane - 70% protein & 30% lipid... contains: a. redox proteins* of Electron Transfer Chain b. ATP synthase* c. many carrier proteins: phosphate translocates, ADP/ATP translocases*, pyruvate/H+ symporter d. α-glycerol-P & malate shuttles enzymes e. fatty acid metabolism (β-oxidation) enzymes

porin

is a channel protein

REDOX POTENTIAL

is a measure of tendency of molecular couple (acceptor/donor) to GAIN-LOSE e's - strong reducing agent (electron donor - NADH) has negative - E'o (redox potential) - strong oxidizing agent (electron acceptor - O2) has positive + E'o (redox potential) (how ΔE'o measured - Reference half-cell* & table )

Oxidative phosphorylation -what is it -ETC (AKA) -ultimate acceptor -is composed of how many enzyme complexes and how many mobile carriers

is the process by which ATP is formed as electrons are transferred from NADH or FADH2 to molecular oxygen (O2) by a series of electron carriers. The energy released form the oxidation of glucose, fatty acids, and amino acids is stored as the reduced coenzymes NADH or FADH2. There is a step by step transfer of electrons from NADH or FADH2 to specific protein complexes, which are part of the electron transport chain. The ultimate acceptor of these electrons is O2. The electron-transport chain, also known as respiratory chain, is a series of linked electron carriers that transfer electrons from NADH and FADH2 to molecular oxygen (O2). The respiratory chain is found in the inner mitochondrial membrane and is composed of 3 enzyme complexes and 2 mobile carriers, also known as respiratory assemblies.

mitochondrial origin

likely endosymbiosis

origin of ATP synthase

may be hydrolytic

Mitochondrial Membrane Transport & the Electron Transfer Chain

membranes = impermeant to most everything, esp to H+ membranes themselves have no electrical charge, but instead they separate electrical charges making the membrane an insulator...an insulator that separates electric charges until used is a battery It is very important to remember that while the outer mitochondrial membrane is permable to most molecules of 5,000 daltons MW, the inner mitochondrial membrane is impermeable to ions and small molecules. This structure is very important because the process of electron transport establishes a proton gradient across the mitochondrial membrane, which is then used to synthesize ATP. If the inner mitochondrial membrane was not impermeable to protons, no gradient could be established

Ubiquinone CoQ - semiquinone & hydroquinone

mobile, membrane bound, non-protein hydrogen carriers

shape of ATP synthase

mushroom shaped complex of 2 membrane subunits F1= in mitoplasm F0= in the inner membrane

Iron sulfur proteins FeS

non-heme iron electron carriers (ferrous+2 <--> ferric+3)

what is complex 2?

succinate-Q dehydrogenase or succinate-Q reductase is positioned between complex 1 and complex 3, but is found on the matrix side of the inner mitochondrial membrane and does not span the membrane. Succinate-dehydrogenase is the point of entry into the respiratory chain for electrons from FADH2.