BioChem Mitochondria (21)
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Name three enzymes that donate electrons to the electron transport chain from their flavin (FAD) prosthetic group
- Succinate dehydrogenase
- acyl-CoA dehydrogenase
- glycerol phosphate dehydrogenase.
How many moles of ATP can be produced per mole of NADH oxidized?
How many moles of ATP can be produced per mole of FADH2 oxidized?
In which organelle does oxidative phosphorylation occur?
- the most important function is energy generation in all cells carrying out aerobic metabolism
- it has an inner (larger, very folded) and outer (smaller) membrane
- the space between the outer and inner membrane (enclosed by the outer membrane but OUTSIDE of the inner membrane)
- contains cellular regulators & myokinase
- the compartment enclosed BY the inner membrane
- contains TCA enzymes, fatty acid oxidation enzymes, amino acid oxidation enzymes, pyruvate carboxylase, mtDNA, mtRNA, & ribosomes
How does the lipid to protein ratio of the outer mitochondrial membrane differ from that of the inner mitochondrial membrane?
- outer mitochondrial membrane is ~ half protein and half lipid
- the inner mitochondrial membrane is ~ 80% protein and 20% lipid
- a transmembrane channel-forming protein found embedded in the outer mitochondrial membrane
- it makes the membrane permeable to most small metabolites that are of molecular weight less than 10,000 Da (10 kDa)
the outer mitochondrial membrane ______ serve as a barrier to proton (H+) diffusion
it does NOT serve as a barrier to proton diffusion
True or False: the outer mitochondrial membrane provides a barrier to proton diffusion
- False, the outer mitochondrial membrane IS permeable to protons
- the cell cytosol and the intermembrane space have ~the same concentration of water soluble metabolites and ions
In which cellular compartment is the electron transport chain located?
the inner mitochondrial membrane
What phospholipid found only in the mitochondrial inner membrane enhances the membrane's lack of permeability to protons?
cardiolipin (diphosphatidyl glycerol)
What are three ways in which mitochondrial DNA differs from nuclear DNA?
- mitochondrial DNA is circular (~17 kB)
- inherited maternally with no paternal contribution
- it contains very few/no introns
- 2-10 copies are present (10^3-10^4 PER cell)
- genetic code differs from that of nuclear DNA's
How many genes does mtDNA encode?
- 13 oxidative phosphorylation proteins (of the 70 needed
- 2 rRNAs
- 22 tRNAs
- some genes overlap
What is encoded by the genes in mitochondrial DNA?
- 2 structural rRNA's
- 22 tRNA's needed for mitochondrial protein synthesis
- 13 of the approximately 70 proteins that form the electron transport chain
Where are all the enzymes of the TCA cycle and fatty acid oxidation synthesized?
they're synthesized as precursors in the cytoplasm, then transported into the mitochondrion where they are converted into mature proteins
How do the vast majority of mitochondrial protein come about?
- most are encoded in the nucleus, synthesized in the cytoplasm, and from there translocated into the organelle
- this includes 80% of the ETC apparatus, all TCA cycle enzymes & fatty acid oxidation enzymes
presequence or matrix targeting sequence
- a sequence of amino acids on the N-terminal of some precursor proteins that directs these proteins to the mitochondria
- usually removed by a processing protease once they're inside the mitochondrial matrix
True or False, mitochondrial protein import is an energy-DEPENDENT (takes energy) process and requires protein unfolding and refolding
translocases of the outer membrane
Which TOM protein recognizes a mt proteins targeting sequence?
translocases of the inner membrane
What did the MOM19 deficiency experiment in yeast show?
- MOM19 were yeast TOM (outer membrane transport proteins)
- without the ability to transport proteins into the organelle, the mitochondria lost it's inner membrane (cristae folds) and died soon after import stopped
What is one reason protein transport into the mitochondria involves energy?
folded proteins can be moved through TOM & TIM channelschaperone activity is required to unfold the proteins so they can move through channels and THIS TAKES ATP
Can ATP come from the diet?
NO - every cell needs to make its own ATP
The function of the respiratory chain is to:
transfer a pair of electrons from NADH (the initial substrate) TO oxygen (the final electron acceptor)
organic molecules associated with different parts of the respiratory chain; they harbor the transferable electrons and mediate electron transfer through the chain
How can electron transport through the respiratory chain to O2 be monitored?
- this electron transport through the respiratory chain can be monitored by oxygen consumption
- remember, even though oxidative phosphorylation (ATP made from ADP + Pi using ATP synthase) is normally tightly coupled to respiration, the two processes ARE separate
- proteins that contain FAD, FMN (flavin mononucleotide), or other flavin derivates as a cofactor
- if the flavin moiety is firmly associated with the enzyme then it's a coFACTOR
- if the flavin moiety can be easily removed it's a coenzyme
Why are electron readily transferred from NADH to flavoproteins?
because flavorproteins usually have a more positive reduction potential than NAD+/NADH
Coenzyme Q (ubiquinone)
molecule that catalyzes the transfer of electrons from Complex I & Complex II to Complex III
Name the four complexes and two transport molecules of the electron transport chain
- Complex I: NADH, CoQ Reductase
- Complex II: succinate, CoQ reductase
- Complex III: reduced CoQ, Cyt C Reductase
- Complex IV: Cytochrome Oxidase
- transport molecules: cytochrome C & ubiquinone (coenzyme Q)
- catalyzes two simultaneous and obligately coupled processes
- 1) the exergonic transfer of a hydride ion from NADH (H-) and a proton (H+) from the matrix to ubiquinone
- NADH + H+ + Q --> NAD+ + QH2
- 2) the endergonic transfer of four protons from the matrix to the intermembrane space
- complex I IS a proton pump
Which complexes of the electron transport chain directly accept electrons from NADH?
Complex I only
Which complexes of the electron transport chain contain the cofactor flavin mononucleotide (FMN)?
Complex I only
Complex II (succinate dehydrogenase)
- it oxidizes succinate --> fumarate and reduces ubiquinone (forming QH2)
- the only enzyme part of both the citric acid cycle & the ETC
- consists of four protein subunits, an FAD cofactor, ironsulfur clusters, & heme group
Does Complex II transport protons across the membrane?
NO complex II does NOT transport protons across the membrane and therefore does NOT contribute to the proton gradient because the reaction it catalyzes (succinate --> fumarate) releases less energy than the oxidation of NADH
Which complexes of the electron transport chain contain the cofactor flavin adenine dinucleotide (FAD)?
Complex II only
What is the role of coenzyme Q (ubiQuinone) in the ETC?
it transfers electrons from Complex I & Complex II to Complex III
- couples the transfer of electrons from ubiquinol (QH2) to cytochrome c with the transport of protons from the matrix to the intermembrane space
- the complex contains cytochrome c1 & two cytochrome b's whose iron atoms alternate between a reduced (+2) and oxidized (+3) ferric state as electrons are transferred through the protein
The reaction catalyzed by complex III is the ________ of one molecule of ubiquinol and the _________ of two molecules of cytochrome c.
- The reaction catalyzed by complex III is the OXIDATION of one molecule of ubiquinol and the REDUCTION of two molecules of cytochrome c
- coenzyme Q can carry two e-, cytochrome c carries only 1 e-
- mitochondrial cytochromes are proteins containing the prosthetic group heme that function in reversible oxidation/reduction reactions
- unlike in RBCs, cytochrome heme doesn't bind oxygen because the cytochrome proteins occupy the space where oxygen would normally bind
- component of complex III & has the most negative reduction potential
- it accepts electrons from reduced coenzyme Q (ubiquinone) CoQH2
- the protons from CoQH2 are released into the intermembrane space (from the matrix)
- component of complex III & has the 2nd most negative reduction potential and transfers an electron from cytochrome b (taken from ubiquinol) to cytochrome c
- (from Complex III cytochrome c moves to Complex IV to donate its electron to a binuclear copper center)
Which molecule catalyzes the transfer of electrons from Complex III to Complex IV?
- mediates the final reaction in the electron transport chain: oxidation of cytochrome c and the reduction of oxygen
- aka transfers electrons to oxygen (reducing it to H2O) while pumping protons across the membrane
- enzyme is made up of 13 subunits, 2 heme groups, copper, magnesium & zinc
What are two ways the reduction of oxygen (final step of the ETC) contribute to creation of the proton gradient?
- 1) provides energy to directly pump protons from the matrix into the intermembrane space
- 2) consumes MATRIX protons when reduced to water (O2 --> 2H2O)
Which complexes of the electron transport chain contain the transition metal copper?
Complex IV only
Which complexes of the ETC contain iron-sulfur (non-heme iron) prosthetic groups?
Complex I, II and III
What is the role of mitochondrial cytochromes in the electron transport chain?
- they transfer electrons sequentially from coenzyme Q to an oxygen molecule
- [ubiQuinon/coenzyme Q] --> cytochrome b --> cytochrome c1 --> cytochrome c --> [oxygen]
Which prosthetic group is common to all mitochondrial cytochromes?
The reduction potential of cytochromes in the electron transport chain ___________ from cytochrome b to cytochrome a3
Which cytochrome in the electron transport chain ACCEPTS electrons from coenzyme Q?
Cytochrome b in complex III
Which cytochromes are components of Complex III?
Cytochrome b and cytochrome c1
Which cytochromes are a component of Complex IV?
cytochrome a and cytochrome a3
What is the location of cytochrome c in the mitochondria?
cytochrome c is found on the outer side (facing the intermembrane space) of the inner mitochondrial membrane
How many protons are pumped across the inner mitochondrial membrane during the transfer of two electrons through Complex I?
How many protons are pumped across the inner mitochondrial membrane during the transfer of two electrons through Complex II?
None, complex II does not pump any protons into the intermembrane space
How many protons are pumped across the inner mitochondrial membrane during the transfer of two electrons through Complex III?
How many protons are pumped across the inner mitochondrial membrane during the transfer of two electrons through Complex IV?
What two additive terms does the proton-motive force created by the ETC consist of?
- pH gradient: the mitochondrial matrix being more alkaline/basic than the intramembranous space (lot's of H+)
- membrane potential: the mitochondrial matrix is negatively charged relative to the proton-rich, positively charged intramembranous space
- the gradient is electrical and chemical
Which respiratory chain complex does not participate in the generation of an electrochemical proton transmembrane potential?
How can the respiratory chain be inhibited?
there are extremely effective inhibitors of Complexes I, III, & IV
Inhibiting which ETC complexes prevents the respiratory chain from completely working?
- Complexes III & IV
- inhibitors of these are extremely toxic
Why doesn't inhibiting Complex I effectively stop the respiratory chain from functioning?
- because there are mitochondrial dehydrogenases in addition to succinate dehydrogenase that input electrons into the respiratory chain at the level of ubiquinone, but NOT through Complex II
- meaning the chain can function effectively even if complex I doesn't supply material for Complex II
Other substrates for mitochondrial dehydrogenases pass electrons into the respiratory chain at the level of ubiquinone, but NOT through Complex II. What is the effect of these electron-transferring enzymes?
- acyl-CoA dehydrogenase
- ubiquinone oxidoreductase
- glycerol-3-phosphate dehydrogenase
- well besides putting more electrons into the pathway, the effect is to contribute to the pool of reduced ubiquinone (QH2) that can be re-oxidized by Complex III
Which complex does rotenone inhibit and how?
it binds to Complex I and prevents the reduction of coenzyme Q
Which complex does antimycin inhibit and how?
it binds to Complex III, preventing the transfer of electrons to Complex IV
Which complex do cyanide and azide inihbit and how?
they bind to the ferric (Fe3+) form of cytochrome a3 in Complex IV and in inhibit cellular respiration
Which complex does carbon monoxide inhibit and how?
carbon monoxide inhibits cellular respiration by binding to the ferrous (Fe2+) form of cytochrome a3 of Complex IV
What are the two pathways that allow mitochondria to utilize cytosolic NADH for oxidative phosphorylation?
- 1. malate-aspartate shuttle
- 2. glycerol phosphate shuttle
- these pathways exist because there is no specific transporter for cytosolic NADH (generated by glycolysis) through the INNER mitochondrial membrane
- malate can carry electrons between the cytoplasm and the mitochondria
- in this shuttle cytoplasmic NADH is used to reduce oxaloacetate to malate, which enters the mitochondrion using the alpha-ketoglutarate transporter (as this happens other alpha-ketoglutarate leave the mitochondrion)
- once in the mitochondrion the malate is reoxidized to oxaloacetate generating NADH
- which can now be used for oxidative phosphorylation
What are the 5 prosthetic groups?
glycerol phosphate shuttle
- cytoplasmic NADH is used to reduce
- dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G-3-P)
- G-3-P is reoxidized by a FAD-linked glycerol phosphate dehydrogenase present on the surface of
- the mitochondrial inner membrane, which transfers electrons to ubiquinone
- FADH2 (rather than NADH) is available for
- oxidation, yielding TWO molecules of ATP INSTEAD of three
What is one difference between the malate-aspartate and glycerol phosphate shuttle?
- unlike malate-aspartate shuttle, no compounds, only electrons, are transferred through the mitochondrial inner membrane in the glycerol phosphate shuttle
- also the malate-aspartate shuttle is REVERSIBLE
- the glycerol phosphate shuttle is IRREVERSIBLE
In the glycerol phosphate shuttle, cytoplasmic NADH is used to reduce _______ to _______
dihydroxyacetone-phosphate to glycerol-3-phosphate
Which enzyme in the glycerol phosphate shuttle transfers electrons from glycerol-3-phosphate to coenzyme Q of the electron transport chain?
FAD-linked glycerol phosphate dehydrogenase
Which enzyme couples the dissipation of a proton concentration gradient to the production of ATP in mitochondria?
ATP synthase (also called F0F1 ATPase, H-dependent ATPase, or simply ATPase)
Complex V - ATP Synthase
- enzyme that catalyzes mitochondrial ATP synthesis is catalyzed by the enzyme ATP synthase
- instead of contributing to the formation of the
- proton gradient, ATP synthase dissipates the gradient using the energy to synthesize ATP
- it's located in the inner mitochondrial membrane
- part of ATP synthase that contains the catalytic site for ATP synthesis and protrudes from the inner mitochondrial membrane into the matrix
- five different hydrophilic subunits that undergo conformational changes to bind ADP + Pi & release ATP
- is where the synthesis of ATP from ADP and Pi occurs
- part of ATP synthase that forms a transmembrane channerl/transmembrane pore that allows protons to flow across the inner mitochondrial membrane
- consists of three different subunits: a, b & c
- protons enter channel and pass through by
- binding to a succession of acidic amino acids
What is the major limiting factor controlling the rate of both respiration and oxidative phosphorylation under normal physiologic conditions?
the availability of ADP
What effect do uncouplers have on the rate of respiration and oxidative phosphorylation?
- uncouplers cause respiration to proceed at maximal rate, but without the production of ATP
- they disrupt the interdependence of respiration and ATP synthesis by allowing protons to bypass the ATP synthase
What mitochondrial enzyme is inhibited by oligomycin?
What is thermogenin?
It's an uncoupling protein found in the mitochondria of brown adipose tissue involved in the generation of heat (non-shivering thermogenesis)
Uncoupler-stimulated respiration releases free energy as:
- regulated proteins with uncoupler (protonophore) function exist in the mitochondrial inner membrane in specialized thermogenic tissues, most notably brown fat
Which two transporters permit the transport of ATP, ADP and phosphate across the inner mitochondrial membrane?
the Pi/OH exchanger and the ATP/ADP exchanger
Name two respiratory toxins that inhibit the inner mitochondrial membrane ATP/ADP exchanger:
Atractyloside and bongkrekic acid
How many moles of ATP are produced from the complete oxidation of glucose via glycolysis, the TCA cycle and respiration?
36-38 moles of ATP per mole of glucose
- characterized by acute or subacute adult onset blindness with a rapid loss of central vision
- blindness is caused by optic nerve death
- point mutation (missense) that causes a single amino acid substitution in a peptide of the NADH-coQ Reductase (Complex I)
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