biochem ch 19

cytrochromes

*1 electron carriers *classes a, b, and c (distinguished by their light absorption spectra due to different ring additions) *each in its reduced Fe2+ state has 3 absorption bands in visible range (600nm in a, 560nm in b, and 550nm in c) *heme group of type c is covalently attached

complex II: succinate dehydrogenase

*FAD accepts 2 e- from succinate *electrons are passed one at a time via Fe-S center to ubiquinone (becomes QH2) *does not transport H+ *succinate dehydrogenase is also an enzyme (membrane bound) in the citric acid cycle *smaller/simpler than complex I (5 prosthetic groups and 4 subunits)

electron movement

*carriers in order of increasing reduction potentials (e- tend to flow from carriers of low E to high E) NADH --> Q --> cytochrome b --> cytochrome c1 --> cytochrome c --> cytochrome a --> cytochrome a3 --> O2 *actual order also depends on concentration of reduced and oxidized forms

coenzyme Q (ubiquinone)

*lipid soluble benzoquinone (conjugated dicarbonyl) with a long isoprenoid side chain *accepts 1-2 electrons in membrane associated electron transfer chain *carries both electrons and protons *freely diffusible in lipid bilayer (mobile electron carrier taking e- from complex I and II to complex III)

mitochondrial matrix contains:

*lower proton concentration/higher pH -pyruvate dehydrogenase complex -citric acid cycle enzymes -fatty acid beta oxidation enzymes -amino acid oxidation enzymes -DNA , ribosomes, other enzymes -ATP, ADP, Pi, Mg2+, Ca2+, K+ -many soluble metabolic intermediates

regulation of oxidative phosphorylation

*mostly regulated by substrate availability (ADP, Pi, and NADH) *regulated by inhibitors of F1 (during low oxygen or at low pH: prevents wasteful hydrolysis of ATP) *--> inhibition of OxPhos leads to accumulation of NADH (feedback inhibition goes to PFK-1 in glycolysis)

complex I: ubiquinone oxidoreductase

*one of the largest macromolecular assemblies (over 40 different polypeptides encoded by both mitochondrial and nuclear genes) *NADH binding site on matrix side *noncovalently bound flavin molecules (FMN) accepts 2 e- from NADH *many iron sulfur centers pass 1 e- at a time to ubiquinone

metabolic poisons to determine sequence

*those after the block become reduced and those after become oxidized *rotenone: NADH -->X Q *antimycin A: cyt b --> X cyt c1 *CN- or CO: Cyt (a+a3) --> X O2

for every pair of electrons transferred to O2

4 H+ are pumped out by complex I, 4 H+ by complex III, and 2 H+ by complex IV. vectorial: NADH + 11H+ + 1/2O2 --> NAD+ + 10H+ + H2O

adenine nucleotide translocase

antiporter located in inner membrane. binds ADP in intermembrane space and exchanges it for ATP (moves 4 negative charges out for every three moved in giving matrix net negative charge) *inhibited by atractyloside

The reaction A + B→ C has a ^ G of -20kJ/mol at 25 C. Starting under standard conditions, one can predict that:

at equilibrium, the concentration of C will be much greater than the concentration of A or B

1. respiratory chain

electrons enter first into the chain of electron carriers (come from dehydrogenases that collect electrons from catabolic pathways and funnel them into universal electron acceptors aka: NAD+, NADP+, FMN, or FAD)

glycerol 3-phosphate dehydrogenase

from glycolysis: this enzyme is a flavoprotein in the inner mitochondrial membrane that channels electrons to reduce ubiquinone to QH2

respirasomes

functional combinations of two or more different electron transfer complexes *cardiolipin (lipid in innermitochondrial membrane) is critical to maintenance

Uncoupling of mitochondrial oxidative phosphorylation:

halts mitochondrial ATP formation, but allows continued O₂ consumption

inner mitochondrial membrane

impermeable to small molecules and ions (including H+). contains: -respiratory electron carriers (complexes I-IV) -ADP-ATP translocase -ATP synthase (F0F1) -other specific membrane transporters -cristae increase surface area

glycerol 3-phosphate shuttle

in skeletal muscle and brain: different NADH shuttle to transfer NADH to ubiquinone and thus into complex III (not complex I) 1.5 ATP per electron pair

In the anatomy of the mitochondrion, the compartment inside the inner membrane is termed which of the following?

matrix

complex IV: Cytochrome oxidase

membrane protein with 13 subunits carries electrons from cytochrome c to O2 forming H2O. 3 critical subunits: -subunit II: 2 Cu ions complexed with -SH groups of 2 Cys residues -subunit I: 2 heme groups (a and a3) and Cu ion *4e- reduction of O2 (1e- at a time) --> 4H+ out of matrix and 4 go towards 2H2O

mass action ratio

the product at all the products divided by the products of all the reactants: [ATP]/([ADP][Pi]) *usually very high

Biological oxidation-reduction reactions always involve:

transfer of electrons

(complex III) ubiquinone: cytochrome c oxidoreductase

transfer of electrons from ubiquinol QH2 to cytochrome c with the vectorial transport of protons from the matrix to the intermembrane space *uses 2 e- from ubiquinone to reduce 2 molecules of cytochrome c *has iron-sulfur clusters, cytochrome bs, and cytochrome cs *Q cycle results in 4 H+ coming into the intermembrane space (IMS)

Electrons from complex I are transferred directly to which of the following electron transport components?

ubiquinone

Which of the following is the hydrophobic electron carrier which is mobile within the membrane

ubiquinone

reduced fuels

used to synthesize ATP in mammals. *reduced fuels ex: carbohydrates, lipids, and amino acids *electrons from reduced fuels transferred to NADH and FADH2 (which are used to make ATP in oxidative phosphorylation)

NADH and NADPH

water soluble electron carriers that associate reversibly with dehydrogenases (NADH carries electrons from catabolic reactions to their entry point on respiratory chain) NADPH supplies electrons to anabolic reactions

formation of reactive oxygen species is favored under 2 conditions

1) mitochondria are not making ATP (lack of ADP or lack of O2) --> high proton motive force and high QH2/Q ratio 2) AND high NADH/NAD+ ratio in the matrix *this produces oxidative stress *superoxide dismutase reverts superoxide radical into H2O2

proton gradient was created in one of 3 ways

1. actively transporting H+ across the membrane complex I and IV 2. chemically remove H+ from the matrix (reduction of CoQ and O2) 3. release H+ into the intermembrane space (oxidation of QH2)

order of electron flow

1. complex I gets e- from NADH 2. complex II gets e- from succinate 3. complex I and II transfer e- to ubiquinone 4. complex III carries e- from ubiquinone to cytochrome c 5. complex IV transfers e- from cytochrome c to O2

three types of electron transfers occurring in oxidative phosphorylation

1. direct transfer of electrons as the reduction of Fe3+ to Fe2+ 2. transfer as a hydrogen atom 3. transfer as a hydride anion (2e-)

6 steps of proton driven rotation of c ring

1. proton enters half channel on P side (cytosol) 2. proton binds to Asp residue and displaces Arg to adjacent c subunit 3. Arg rotates, displacing H+ from Asp 4. displaced H+ exits on N side 5. c ring rotates, Arg returns to Pside half channel 6. process repeats

coupling proton translocation to ATP synthesis

1. proton translocation causes rotation of Fo subunit and central shaft (gamma) 2. causes conformational change with all the alpha-beta pairs 3. the conformational change in 1 of the 3 pairs promotes condensation of ADP to Pi

steps of chemiosmotic theory

1. reduced substrate (fuel) donates e- 2. electron carriers pump H+ out as electrons flow to O2 3. energy of e- flow stored as electrochemical potential 4. ATP synthase uses electrochemical potential to synthesize ATP

proton motive force

1. the chemical potential energy created from difference in H+ concentration 2. electrical potential energy that results from the separation of charge (H+ moves across the membrane without a counter ion)

complex I catalyzes two simultaneous coupled processes

1. the exergonic transfer to ubiquinone of a hydride ion from NADH and a proton from the matrix (NADH + H+ + Q --> NAD+ +QH2 2. endergonic transfer of 4 protons from the matrix to the intermembrane space *proton pump driven by energy of electron transfer

mitochondrial genome

16.5k base pairs. only 37 genes (24 encode tRNA, and rRNA) *about 5 copies of genome/mitochondrian *only 1,100 proteins are found in/encoded by mitochondria (thus, majority encoded by nucleus)

summary of electron transport: complex I --> complex IV

1NADH + 11H+ + 1/2O2 --> NAD+ + 10H+ + H2O

chemiosmotic theory

A model to explain the synthesis of ATP. The theory proposes that the energy for ATP synthesis originates from the electrochemical gradient of protons across a membrane (energy by electron transport down gradient drives H+ against gradient) *ADP + Pi =ATP (highly thermodynamically unfavorable) *also H+ down gradient drives phosphorylation of ADP

acceptor control

Regulation of respiration by availability of ADP *acceptor control ratio: ratio of maximal rate of ADP induced O2 consumption to the basal rate in absence of ADP (ratio is at least 10)

Cyanide, oligomycin, and 2,4-dinitrophenol (DNP) are inhibitors of mitochondrial aerobic phosphorylation. Which of the following statements correctly describes the mode of action of the three inhibitors?

Cyanide inhibits the respiratory chain, whereas oligomycin and 2,4 dinitrophenol inhibit the synthesis of ATP

vectorial

Describes specific directional transfer of protons across the Mt inner membrane *inner membrane space becomes + charged and matrix becomes - charged

ATP synthase

F-type ATPase in inner mitochondrial membrane that catalyzes formation of ATP from ADP and Pi. Driven by flow of H+ from p to n side of membrane.

2 distinct components of ATP synthase

F1: peripheral membrane protein Fo: integral membrane (transports protons from IMS to matrix to dissipate gradient; energy transferred to F1 to catalyze phosphorylation of ADP)

summary of electron transport: complex II --> complex IV

FADH2 + 6H+ + 1/2O2 --> FAD + 6H+ + H2O

iron-sulfur proteins

Fe-S centers (iron coordinated with 4 cys residues) with equal number of Fe and S atoms *rieske iron-sulfur protein is 1 Fe coordinated to two His residues instead *1 electron transfer

malate-aspartate shuttle

In heart, muscle, and liver. Transfers electrons from cytosol to mitochondrial NADH. Net effect: NADHp --> NADHn *cytosolic NADH transferred to cytosolic oxaloacetate to make malate --> passes through inner membrane via transporter --> forms NADH *2.5 ATP per electron pair

types of electron carrying molecules

NAD, flavoproteins. and three types of electron carriers in the electron transport chain: 1. ubiquinone (hydrophobic quinone) 2. cytochromes 3. iron-sulfur proteins (last two are iron containing proteins)

binding change model

Proposed mechanism for the action of ATP synthase, involving concerted conformational changes, rotational motions, catalysis, and substrate binding and release. *gamma subunit rotates in one direction when synthesizing ATP and opposite when hydrolyzing

The relative concentrations of ATP and ADP control the cellular rates of

all of the above

poisons that block electron flow from Fe-S centers of complex I to ubiquinone

amytal, rotenone, and piericidin A

Which of the following transfers electrons by diffusion on the outside of the membrane

cytochrome c

acyl CoA dehydrogenase

catalyzes the first step in beta oxidation of fatty acyl CoA. transfers e- to FAD of the dehydrogenase then to ETF then to ubiquinone

Succinate interacts directly with which component of the electron transport components?

complex II

flavoproteins

contain tightly (sometimes covalently) bound flavin nucleotide FMN or FAD. can accept one or two electrons

The cytochrome that passes electrons directly to oxygen is

cytochrome a/a3 complex

The final complex in mitochondrial electron transport chain, which catalyzes the terminal transfer of electrons to molecular oxygen, is termed

cytochrome oxidase

Q cycle

net effect: QH2 is oxidized to Q, 2 molecules of cytochrome c are reduced and protons are moved from the P side to the N side *mediates transfer of electrons from the 2e- carrier to single e- carriers *4 H+ transported per pair of e- that reach CytC (4H+ per reduced enzyme Q)

outer mitochondrial membrane

porous membrane allows passage of metabolites

Fo complex

proton pore. 3 subunits: a, b, c (# of c subunits vary) *c is small, hydrophobic: the c subunits in the c ring rotate together as a unit around an axis perpendicular to the membrane (inner circle is amino terminal helices of each c subunit)

NAD-linked dehydrogenases

remove two H atoms from their substrates. 1 is transferred as a hydride ion H- to NAD+. the other is released as H+ in the medium

inner membrane space

similar environment to cytosol, higher proton concentration/lower pH

cytochrome c

soluble protein of intermembrane space (IMS) . its heme accepts e- from complex III and moves to the copper on complex IV

F1

soluble, individually catalyzes hydrolysis of ATP, hexamer arranged in three alpha-beta dimers. each beta subunit can exist in 3 formations: -open: empty -loose: binding ADP to Pi -tight: catalyzes ATP formation and binds product

reactive oxygen species (ROS)

superoxide free radical is generated when the Q- radical (intermediate) is passed to O2 instead of QH2. (leads to hydroxyl free radical) *more common when flow of electrons through respiratory chain is slowed

phosphate translocase

symporter that brings in Pi (H2PO4) along with one proton into the matrix. driven by the PMF.