Biochemistry Final Exam (Exam 4) F2020
Proton Transport by Cytochrome c (CIV) Oxidase
Four protons are taken up from the matrix side to reduce one molecule of O2 to two molecules of H2O. • These protons are called "chemical protons" because they participate in a clearly defined reaction with O2. (reduction to make H2O) • Four additional "pumped" protons are transported out of the matrix and released on the cytoplasmic side in the course of the reaction. • The pumped protons double the efficiency of free-energy storage in the form of a proton gradient for this final step in the electron- transport chain. shuffle e- to reduce O2 to H2O and create proton gradient to power the synthesis of ATP
Iron-sulfur Clusters are Common Components of the Electron Transport Chain
Iron-sulfur proteins, also called nonheme iron proteins, are also prominent electron carriers. • These proteins contain various types of iron-sulfur clusters. Like cytochromes, the iron cycles between Fe2+ and Fe3+ (different oxidation states) as it accepts or donates electrons. • Frataxin is a small mitochondrial protein that is crucial for the synthesis of Fe-S clusters. Deficiency in frataxin results in Friedreich's ataxia, which affects the nervous system as well as the heart and skeletal systems.
Rotational Catalysis is the World's Smallest Molecular Motor
It is possible to observe the rotation of the γ subunit directly. • Cloned α3β3γ subunits were attached to a glass slide that allowed the movement of the γ subunit to be visualized as a result of ATP hydrolysis. • The hydrolysis of a single ATP powered the rotation of the γ subunit 120°
Structure of ATP Synthase
A schematic structure is shown along with representations of the components for which structures have been determined to high resolution. • The P-loop NTPase domains of the α and β subunits are indicated by purple shading. Notice that part of the enzyme complex is embedded in the inner mitochondrial membrane, whereas the remainder resides in the matrix. Big R shape that closes around the membrane
Malate-aspartate Shuttle
NADH--NAD+ oxidized oxaloacetate to malate to oxaloacetate using NAD+ to NADH
Iron-Sulfur Clusters
(A) A single iron ion bound by four cysteine residues. (B) 2Fe-2S cluster with iron ions bridged by sulfide ions. (C) 4Fe-4S cluster. Each of these clusters can undergo oxidation-reduction reactions. Fe-undergoes redox -sulfur from cysteine (amino acid residues from protein complex) -all iron ions are attached via a SULFUR BRIDGE
A Dimer of Mitochondria ATP Synthase
A schematic representation of a dimer of mitochondria ATP synthase is shown embedded in the inner mitochondrial membrane. • The dimer, joined by an assortment of proteins, assists in bending the inner mitochondria membrane. -cristae: INCREASES surface area of inner membrane, increases e transport chain and ATP synthase • The structure of the dimer was determined by cryo- electron microscopy.
peroxide that is part of CIV peroxide bridge
A3--O--O--CuB a3 with Fe at center O--O is peroxide bridge CuB with Cu at center
18.3 the respiratory chain consists of 4 Complexes: Three Proton Pumps and a Physical Link to the Citric Acid Cycle
ADP---ATP not spontaneous on own, but is coupled Electrons flow from NADH to O2 through three large protein complexes embedded in the inner mitochondria membrane. • These complexes pump protons out of the mitochondrial matrix, generating a proton gradient. Proton movement back in through ATP synthase powers substantial ATP synthesis (pulls protons back in). • The complexes are: - NADH-Q oxidoreductase (Complex I). (NADH through this) - Q-cytochrome c oxidoreductase (Complex III). - -Cytochrome c oxidase (Complex IV). --all proton pumps • An additional complex, succinate Q-reductase (Complex II), delivers electrons from FADH2 to Complex III. - Succinate-Q reductase is NOT a proton pump CI--Q (CII to Q)--III--Cyto c--IV
ATP Synthase and G Proteins Have Several Common Features
ATP synthase and the Gα subunit of heterotrimeric G proteins are members of the P-loop NTPase family of proteins. • They do not exchange nucleotides unless they are stimulated to do so by interaction with other proteins.
Proton Flow through ATP Synthase Leads to the Release of Tightly Bound ATP: The Binding-change Mechanism ATP and ADP must bind to ____ to function as substrates the reaction proceeds through a _______ intermediate
ATP synthase catalyzes the formation of ATP from ADP and Pi . • ATP and ADP must bind to Mg2+ to function as substrates. • The reaction proceeds through a pentacovalent intermediate. (phosphorous with 5 bonds)
ATP Synthase is Composed of a Protonconducting Unit and a Catalytic Unit
ATP synthase is made up of two components: o The F1 component contains the three active sites, located on the three β subunits, and protrudes into the mitochondrial matrix. o The F0 component is embedded in the inner mitochondrial membrane and contains the proton channel. • The γ subunit connects the F1 and F0 components. • Each β subunit is distinct in that each subunit interacts differently with the γ subunit. • The association of synthases with one another facilitates the formation of cristae. (the folds of the inner mitochondrial membrane)
physical link to the citric acid cycle
complex II
electron transport chain purpose
create a proton gradient in order to have ATP synthesis
The Respirasome
cyt c bound to CIII The structure of the electrontransport chain supercomplex or respirasome has been determined by cryo-electron microscopy . • The complex consists of two copies each of Complex I, Complex III, and Complex IV. Top and side views are shown, with cytochrome c bound to Complex III.
Evolutionary Tree Constructed from Sequences of Cytochrome c
Branch lengths are proportional to the number of amino acid changes that are believed to have occurred. humans and monkeys are very similar *** been around for a long time-critically important for all species
respiration two parts e- donor and acceptor
Collectively, the citric acid cycle and oxidative phosphorylation are called cellular respiration or simply respiration. • Respiration is defined as an ATP-generating process in which an inorganic compound (e.g., O2-molecular oxygen) serves as the ultimate electron acceptor. The electron donor can be either an organic or inorganic compound.
The Conformation of Cytochrome c Has Remained Essentially Constant for More than a Billion Years
Cytochrome c is a highly conserved protein. • Cytochrome c from any eukaryotic species will react in vitro with the cytochrome c oxidase from any other species. (ANY cyto c and cyto c oxidase can work together • Examination of the primary structure of cytochrome c from a variety of species allowed the construction of phylogenetic trees.
Cytochrome c Oxidase Catalyzes the Reduction of Molecular Oxygen to Water
Cytochrome c oxidase (C IV) accepts four electrons from four molecules of cytochrome c in order to catalyze the reduction of O2 to two molecules of H2O • In the cytochrome c oxidase reaction, eight protons are removed from the matrix. Four protons, called chemical protons, are used to reduce oxygen. In addition, four protons are pumped into the intermembrane space 4 to make H2O and 4 for the proton pump 4Cytc red + 8H+ matrix + O2 --- 4 Cyt c ox + 2H2O (end goal of e- T C) + 4H+ intermembrane space (pumping out protons-proton gradient powers ATP synthesis)
Cytochrome c Oxidase (C IV) Catalyzes the Reduction of Molecular Oxygen to Water
Cytochrome c oxidase consists of 13 subunits. The enzyme requires two heme A moieties, designated heme a and heme a3, and two copper centers. (accepts e- from cyt c CuA-CuA) Copper center A contains two copper ions, whereas center B is coordinated to three histidine residues, one of which is covalently linked to tyrosine. • Two electrons flow according to the pattern: cytochrome c à CuA/CuA à heme a à heme a3 à CuB. • When the Fe of heme a3 and CuB are reduced, they bind oxygen as a peroxide bridge between them. A3--O--O--CuB peroxide that is part of CIV • The addition of two more electrons and four protons (chemical; protons and other 4 are pumped out) generates two molecules of water
CH 18 objectives
Describe the key components of the electron-transport chain and how they are arranged. 2. Explain the benefits of having the electron-transport chain located in a membrane. 3. Describe how the proton-motive force is converted into ATP. 4. Identify the ultimate determinant of the rate of cellular respiration.
Chemiosmotic Hypothesis
Electron transfer through the respiratory chain leads to the pumping of protons from the matrix to the cytoplasmic side of the inner mitochondrial membrane. • The pH gradient and membrane potential constitute a proton- motive force that is used to drive ATP synthesis.
The Malate-aspartate Shuttle
In heart and liver, electrons from cytoplasmic NADH are used to generate mitochondrial NADH in the malate-aspartate shuttle. • The malate-aspartate shuttle consists of two membrane transporters and four enzymes. -get NADH into the mitochondria -NADH from cytoplasm into the mitochondria and NAD+ from mitochondria into NADH in mitochondria
Site of oxidative phosphorylation
Mitochondria, stained green, form a network inside a fibroblast cell (left) • Mitochondria oxidize carbon fuels to form cellular energy in the form of ATP.
Electron Carrier Oxidation Can Power ADP Rephosphorylation
NADH oxidation is exceptionally exergonic: • ADP rephosphorylation is only quite endergonic: -220 • Proton-motive force is used to drive ATP synthesis: +30
Progressive Alteration of the Forms of the Three Active Sites of ATP Synthase
No two subunits are ever in the same conformation. • Each subunit cycles through the three conformations. (ex: never two in O form) • The movement of the γ subunit in response to proton flow powers the interconversion of the forms. (B1, B2, B3 can all cycle through O,L, and T)
Conservation of the Three-dimensional Structure of Cytochrome c
Notice the overall structural similarity of the different molecules from different sources. • The side chains are shown for the 21 conserved amino acids as well as the centrally located planar heme similar structures
18.5 Many Shuttles Allow Movement Across Mitochondrial Membranes one function of the respiratory chain is to regenerate ______ for use in glycolysis but the inner mitochondrial membrane is impermeable to _____ and ____ solution in muscle
One function of the respiratory chain is to regenerate NAD+ (required for reduction of DHAP to GAP) for use in glycolysis, but the inner mitochondrial membrane is impermeable to NADH and NAD+. • A solution: in muscle, electrons from cytoplasmic NADH can enter the electron-transport chain using the glycerol 3-phosphate shuttle. • In that example, the electrons are transferred from NADH to FADH2 and subsequently to Q to form QH2. • When cytoplasmic NADH transported by this shuttle is oxidized by the respiratory chain, 1.5 rather than 2.5 molecules of ATP are formed, because FAD rather than NAD+ is the electron acceptor. in muscles
Overview of Oxidative Phosphorylation
Oxidation and ATP synthesis are coupled by transmembrane proton fluxes. Electrons flow from NADH and FADH2 through four protein complexes to reduce oxygen to water (yellow tube). --O2 reduced to H2O with 4 different complexes--the electron transport chain happens here, protons pumped out • Three of the complexes pump protons from the mitochondrial matrix to the exterior of the mitochondria. The protons return to the matrix by flowing through another protein complex, ATP synthase (red structure), powering the synthesis of ATP. --ATP synthase and protons in
Oxidative Phosphorylation
Oxidative phosphorylation captures the energy of highenergy electrons to synthesize ATP in mitochondria (powerhouse) • The flow of electrons from NADH and FADH2 to O2 occurs in the electron-transport chain or respiratory chain. • This exergonic set of oxidation-reduction reactions generates a proton gradient, and then the proton gradient is used to power the synthesis of ATP.
Toxic Derivatives of Molecular Oxygen such as Superoxide Radicals Sre Scavenged by Protective Enzymes
Partial reduction of O2 generates highly reactive oxygen derivatives, called reactive oxygen species (ROS). • ROS are implicated in many pathological conditions. • ROS include superoxide ion (O2- radical), peroxide ion (O2 2-), and hydroxyl radical (OHradical). O2 ---lose e----superperoxide ion--lose e---O2 2- peroxide enzymes take care of super reactive byproducts
Mechanism of Mitochondrial ATPADP Translocase
high concentration of ADP bind and flip into matrix -then open to bind ATP and flip back into cytoplasm -takes 25% of the proton motive force
Components of the ElectronTransport Chain
in intermembrane space -from high to low energy--spontaneous complex -e- flow down a gradient from NADH to O2 (downhill in energy) -the flow is catalyzed by 4 protein complexes, and the energy released is used to generate a proton gradient -point of this gradient is to help create ATP b/c cannot occur on its own -shuffle e- from C1 from NADH to Q and II e- to Q then to III to cytochrome c to IV--power reduction of O2 to H2O
Testing the Chemiosmotic Hypothesis
it has been tested and proven ATP is synthesized when reconstituted membrane vesicles containing bacteriorhodopsin (a lightdriven proton pump) and ATP synthase are illuminated.
Proton Motion across the Membrane Drives Rotation of the c Ring
one 1/2 channel open in intermembrnae space with lots of H+ when protons come in, bind to aspartate or glutamate and power rotation so H+ goes into other 1/2 channel to other 1/2 channel and into the matrix
Pathological Conditions that May Entail Free-radical Injury
parkinson's, downs syndrome, renal failure, etc.
Components of the Mitochondrial Electron-transport Chain most components have _____ ____ which are required for full catalytic activity -most common ones are
prosethetic groups (most have iron)-coenzymes or metals required for full catalytic activity -iron, sulfur, heme (iron at center)
Distance Dependence of Electrontransfer Rate
the faster the e- is moving, the smaller the distance it travels -proteins are like crossing guards and give e- a greater chance of getting to correct location -proteins facilitate movement of directions
why do we need to use the redox processes?
they are spontaneous (-220 KJ/mol) and adding a P to ADP is not (+30 kJ/mol) -trying to get e- from C1 and C2 through to CIV b/c trying to make ATP and need energy from the proton gradient requires the e- transport chain
transmembrane helices of Q-cytochrome c Oxidoreductase
tubes/barrels are embedded in bilayer (proton pump) -in phospholipid bilayer
inner membrane of mitochondria ridges site of what?
• The inner membrane, which is folded into ridges called cristae, is impermeable to most molecules. -inter-membrane space • The inner membrane is the site of electron transport and ATP synthesis.
The Q Cycle Funnels Electrons from a Two electron Carrier (QH2) to a One-electron Carrier and Pumps Protons
-QH2 from Q carries two electrons, whereas cytochrome c carries only one electron. • The mechanism for coupling electron transfer from QH2 to cytochrome c is called the Q cycle. In the first half of the Q cycle, one electron from QH2 reduces cytochrome c and one reacts with Q to form Q −. (semi-quinone radical anion) In the second half of the cycle, another QH2 reduces cytochrome c and Q −. • In one Q cycle (both halves of the cycle), four protons are pumped out of the mitochondrial matrix and two additional ones are removed from the matrix 2QH2 + Q + 2cytc (oxidized) + 2H+ (pull two from matrix) --- 2Q + QH2 + 2 cytc (reduced) + 4H+ (pump out 4 to intermembrane space) first half: QH2 from Q pool which came from Q (e- from NADH (from CAC to FMN to Iron-Sulfur clusters to Q) and FADH2 reduced to QH2) -cytochrome c can only accept one e- at a time from QH2 which has 2 -1 electron to cytochrome c1 then to cyt C (QH2 to Q) and other e- to cytochrome b to create semiquinone radical anion second half: needed two e- to fully reduce cytochrome c, but can only take one at a time -QH2 from Q pool passes e- to cytochrome c1 to cytochrome c and cytochrome c is fully reduced now -other e- from QH2 to semiquinone radical anion and input of protons gives QH2 which becomes part of the Q pool
2 e- points
2 e- entry points: NADH to CI and FADH to CII--both shuffle e- to Q to be reduced to QH2
Most of the Electron-transport Chain is Organized into a Complex Called the Respirasome
Although the electron transport chain is often depicted as individual units, research suggests that three of the components are arranged in a large complex known as the respirasome. • The human respirasome is believed to consist of two copies each of Complex I and Complex IV surrounding two copies of Complex III. o Also, two copies of cytochrome c are located on the surface of Complex III. o Although not experimentally established to date, the structure allows for Complex II (Link to c acid cycle--e- from complex II to Q ubiquinone) to associate in a gap between Complexes I and IV
Electrons Can Be Transferred between Groups that Are Not in Contact
Electrons can move through space, even through a vacuum, although the rate of electron transfer through space falls off rapidly as the electron donor and electron acceptor move apart from each other. • Proteins provide an environment that allows efficient transfer of electrons.
Coupled Electron-proton Transfer Reactions through NADH-Q Oxidoreductase
Electrons flow in Complex I from NADH through FMN and a series of iron-sulfur clusters to ubiquinone (Q), forming Q2−. The charges on Q2− are electrostatically transmitted to hydrophilic amino acid residues [shown as red (glutamate) and blue (lysine or histidine) balls] that power the movement of HL and βH components. This movement changes the conformation of the transmembrane helices and results in the transport of four protons out of the mitochondrial matrix. -as e- slow, conformational changes in proteins allow for protons to be pumped out
Glycerol 3-phosphate Shuttle
Electrons from NADH can enter the mitochondrial electron-transport chain by being used to reduce dihydroxyacetone phosphate to glycerol 3-phosphate. • Glycerol 3-phosphate is reoxidized by electron transfer to an FAD prosthetic group in a membrane-bound glycerol 3-phosphate dehydrogenase. • Subsequent electron transfer to Q to form QH2 allows these electrons to enter the electron-transport chain. as DHAP is reduced to GAP, NADH oxidized to NAD+ then GAP oxidation to DHAP b/c reduction of FAD---FADH2 to get e- into the electron transport chain
Electrons Flow from Ubiquinol to Cytochrome c through Q-cytochrome c Oxidoreductase (complex III) e- from ____ are used to reduce two molecules of ______ ___ in a reaction catalyzed by ___ ______ ___ _____ complex III has two types of _____ named __ and ____
Electrons from QH2 are used to reduce two molecules of cytochrome c in a reaction catalyzed by the Qcytochrome c oxidoreductase, or Complex III. Complex III is also a proton pump. • Complex III contains two types of cytochromes named b and c1. -through CIII to get to cytochrome c, but within CIII are cytochrome b and c1 QH2 + 2Cytc (oxidized) + 2H+ matrix---Q (reform Q) + 2cytc (reduced) + 4H+ (intermembrane space)
Ch 18 outline
Eukaryotic Oxidative Phosphorylation Takes Place in Mitochondria 2) Oxidative Phosphorylation Depends on Electron Transfer 3) The Respiratory Chain Consists of Four Complexes: Three Proton Pumps and a Physical Link to the Citric Acid Cycle 4) A Proton Gradient Powers the Synthesis of ATP 5) Many Shuttles Allow Movement Across Mitochondrial Membranes 6) The Regulation of Cellular Respiration Is Governed Primarily by the Need for ATP
e- from NADH to
FMN--iron-sulfur clusters--Q
Oxidation States of Flavins
FMN-flavin mononucleotide oxidized has a phosphorylated sugar molecule -add 2e- and 2H+ to flavin mononucleotide reduced-FMNH2 (removing double bonds and reducing Hs) with Q, 2 ketones reduced to 2 alcohols FMN is the first e- acceptor of NADH
Heme A
Fe coordinated in middle
Frataxin
Frataxin is a small mitochondrial protein that is crucial for the synthesis of Fe-S clusters. Deficiency in frataxin results in Friedreich's ataxia, which affects the nervous system as well as the heart and skeletal systems. -crucial for Fe-S clusters--crucial for e- transport chain--crucial for proton gradient--crucial for ATP synthesis and muscle contraction
The Electron Transport Chain
High-energy electrons in the form of NADH and FADH2 are generated by the citric acid cycle. • These electrons flow through the respiratory chain, which powers proton pumping and results in the reduction of O2 tpo H2O
Proton Flow around the c Ring Powers ATP Synthesis
Proton flow occurs through the Fo component of the ATP synthase. • Subunit a, which abuts the c ring, has two channels that reach halfway into the a subunit. One half-channel opens to the intermembrane space and the other to the matrix. • Protons enter the half-channel facing the proton-rich intermembrane space, bind to a glutamate or aspartate residue on one of the subunits of the c ring, and then leave the c subunit once it rotates around to face the matrix half channel. protons bind one half channel, rotate, move through other channel and then release • The force of the proton gradient powers rotation of the c ring b/w the 1/2 channels • The rotation of the c rings powers the movement of the γ subunit, which in turn alters the conformation of the β subunits.
Mitochondria Are the Result of an Endosymbiotic Event
Sequence data suggest that all mitochondria are descendants of an ancestor of Rickettsia prowazekii, which was engulfed by another cell. • The most bacteria-like mitochondrial genome is that of the protozoan Reclinomonas americana. The genome of R. americana encodes less than 2% of the proteinencoding genes of E. coli. • All mitochondrial genomes have approximately the same 2% of bacterial genes, suggesting that an endosymbiotic event occurred just ONCE in evolution
Ubiquinol Is the Entry Point for Electrons from FADH2 of Flavoproteins
Succinate dehydrogenase of the citric acid cycle is a part of the succinate-Q reductase complex (Complex II). • The FADH2 generated in the citric acid cycle reduces Q to QH2, which then enters the Q pool. • Complex II is not a proton pump--it is the physical link to the citric acid cycle
Superoxide Dismutase
Superoxide dismutase (SOD) and catalase help protect against ROS damage. (both protect from damage from radical oxygen species) 2O2- radical + 2H+ ------ superoxide dismutase----O2 + H2O2 o Eukaryotes express two forms of this enzyme, a manganese-containing mitochondrial form and a copper-and-zinc-dependent cytoplasmic form. o Exercise is associated with increased SOD expression. --with sufficient O2, going through e- transport chain to create ATP also create reactive O, so need to balance out • The hydrogen peroxide that is produced by SOD is then scavenged by catalase
Mitochondrial Transporters for Metabolites Have a Common Tripartite Structure
The ATP-ADP translocase is composed of three tandem repeats of a 100-amino acid domain, with each domain containing two transmembrane segments. • In addition to the translocase, the inner mitochondrial membrane has many transporters, or carriers, to enable the exchange of ions or charged molecules between the mitochondrial matrix and cytoplasm
The Entry of ADP into Mitochondria Is Coupled to the Exit of ATP by ATP-ADP Translocase
The ATP-ADP translocase, which constitutes 15% of the protein of the inner mitochondrial membrane, enables the exchange of cytoplasmic ADP for mitochondrial ATP. • ADP must enter the mitochondria for ATP to leave. • Neither ATP nor ADP is bound to Mg2+. ((MUST BE BOUND to Mg2+ to be a substrate)) • The ATP-ADP exchange is energetically expensive. Approximately 25% of the proton-motive force generated by the respiratory chain is consumed by this exchange process ADP from cytoplam into the matrix and ATP from matrix into the cytoplasm
Proton Flow through the ATP Synthase Allows Release of the Newly Synthesized ATP
The binding-change mechanism accounts for the synthesis of ATP in response to proton flow. • The three catalytic β subunits of the F1 component (out into the mitochondrial matrix) can exist in three conformations: o In the O (open) form, nucleotides can bind to or be released from the β subunit. (bind and release) o In the L (loose) form, nucleotides (ADP with P) are trapped in the β subunit. o In the T (tight) form, ATP is synthesized from ADP and Pi in the absence of a proton gradient but cannot be released from the enzyme. (made but trapped) • Proton flow releases the newly synthesized ATP. (form ATP synthase)
ATP Synthase Nucleotide-binding Sites Are Not Equivalent
The γ subunit passes through the center of the α3β3 hexamer and makes the nucleotide-binding sites in the β subunits distinct from one another. • The β subunits are colored to distinguish them from one another.
Components of the ProtonConducting Unit of ATP Synthase
The c subunit consists of two α helices that span the membrane. In E. coli, an aspartic acid residue in one of the helices lies on the center of the membrane. • The structure of the a subunit has not yet been directly observed, but it appears to include two half- channels that allow protons to enter and pass partway but not completely through the membrane. intermembrane half channel has high [H+] and goes into 1/2 channel, turns, then into matrix 1/2 channel into low [H+]
18.1 Eukaryotic Oxidative Phosphorylation Takes Place in Mitochondria
The electron-transport chain and ATP synthesis occur in the mitochondria. • Recall that the citric acid cycle occurs in the mitochondrial matrix.
18.2 Oxidative Phosphorylation Depends on Electron Transfer
The electron-transport chain is a series of coupled oxidation-reduction (redox) reactions that transfer electrons from NADH and FADH2 to oxygen. -reduction of O2 to H2O
The Electron Transport Chain (1/2) coenzyme Q derived from ____, binds protons (____) and electrons and can exist in several ________ _______ -_______ Q and ______ Q are present in the inner mitochondrial membrane in what is called the ____ pool -cytochrome c is and electron carrier that employs an ____ incorporated into ____ -cytochromes are ____-_____ proteins that contain a ____ prosthetic group
The electrons donated by NADH and FADH2 are passed to electron carriers in the protein complexes. • Coenzyme Q, which is derived from isoprene, binds protons (QH2) as well as electrons and can exist in several oxidation states. • Oxidized (Q) and reduced Q (QH2) are present in the inner mitochondrial membrane in what is called the Q pool. • Cytochrome c (links III to IV) is an electron carrier that employs an iron incorporated into heme. Cytochrome c carries electrons from Complex III to Complex IV. • In general, cytochromes are electron-transferring proteins that contain a heme prosthetic group. The heme iron cycles between Fe2+ and Fe3+ as it accepts or donates electrons.
Electron Flow from NADH to Molecular Oxygen Powers the Formation of a Proton Gradient 2.0 -Can the energy associated with the proton gradient be quantified?
The energy associated with a proton gradient can be quantified by using the following equation: ΔG = RT ln (c2 /c1 ) + ZFΔV JUST KNOW: the energy associated with the proton gradient can be quantified where c1 is the concentration of the protons on one side of the membrane and c2 is the concentration of protons on the side of the gradient to which the protons are moving, Z is the charge on the proton, ΔV is the voltage potential across the membrane, R is the gas constant, and T is the temperature in kelvin
Coupling of Electron Carrier Oxidation and ADP Phosphorylation
The flow of electrons from reduced carriers such as NADH (NADH and FADH2)is highly exergonic--spontaneous. NADH + 1/2 O2 + H+ ---- H2O + NAD+ change in G knot prime=-220.1 kj/mol • COUPLED WITH REDUCTION in order to power ATP synthesis This is sufficient to power the rephosphorylation of multiple copies of ADP. • ADP + Pi + H+ -----ATP + H2O change in G knot prime=+30.5 kj/mol The coupling of electron carrier oxidation with ADP rephosphorylation occurs with the participation of the inner mitochondrial membrane.
Overlapping Gene Complements of Mitochondria -what are mitochondria derived from?
The genes present within each oval are those present within the organism represented by the oval. Only rRNA- and protein-coding genes are shown. • The genome of Reclinomonas contains all the protein-coding genes found in all the sequenced mitochondrial genomes only rRNA and protein coding genes MAIN POINT: derived from bacteria, incorporated into cell and 2% of coding region present in eukaryotic cells
Mitochondria Are Bounded by a Double Membrane -outer membrane is _____ because of _____ inner membrane is _______ into _____ is __________ site of _______ _______ and ______ ________ which two processes occur in the matrix?
The outer mitochondrial membrane is permeable to most small ions and molecules because of the channel protein mitochondrial PORIN. • The inner membrane, which is folded into ridges called cristae, is impermeable to most molecules. • The inner membrane is the site of electron transport and ATP synthesis. • The citric acid cycle and fatty acid oxidation occur in the matrix.
Superoxide Dismutase Mechanism
The oxidized form of superoxide dismutase (Mox) reacts with one superoxide ion to form O2 and generate the reduced form of the enzyme (Mred). • The reduced form then reacts with a second superoxide and two protons to form hydrogen peroxide and regenerate the oxidized form of the enzyme.
18.4 A Proton Gradient Powers the Synthesis of ATP
The proton gradient generated by the oxidation of NADH and FADH2 is called the proton-motive force. • The proton-motive force powers the synthesis of ATP. • The proton-motive force consists of a chemical gradient and a charge gradient. • Heterologous experimental systems confirmed that proton gradients can power ATP synthesis.
The Electron-transfer Potential of an Electron is Measured as Redox Potential what is E0'
The reduction potential E0', or redox potential, is the measure of a molecule's tendency to donate or accept electrons. • A strong reducing agent readily donates electrons and has a negative E0' , while a strong oxidizing agent readily accepts electrons and has a positive E0'. • The standard free-energy change is related to the change in reduction potential: • where n is the number of electrons transferred and F is the Faraday constant. E0' is the ability to donate or accept electrons -E0' donating e- +E0' is accepting e-
ATP Forms without a Proton-motive Force but Is Not Released
The results of isotopic-exchange experiments indicate that enzyme-bound ATP is formed from ADP and Pi in the absence of a proton-motive force.
Binding-change Mechanism for ATP Synthase
The rotation of the γ subunit interconverts the three β subunits. The subunit in the T (tight) form interconverts ADP and Pi and ATP but does not allow ATP to be released. • When the γ subunit (powered by proton motive force b/c F0 is the proton pump that is connected to y) is rotated by 120 degrees in a counterclockwise (CCW) direction, the T-form subunit is converted into the O form, allowing ATP release. • ADP and Pi can then bind to the O-form subunit. An additional 120- degree rotation (not shown) traps these substrates in an L-form subunit.
Q Cycle
The shuttling of electrons between ubiquinol and ubiquinone in the inner mitochondrial membrane as a part of Complex III's function
Sizes of Mitochondrial Genomes
The sizes of three mitochondrial genomes compared with the genome of Rickettsia, a relative of the presumed ancestor of all mitochondria. • For genomes of more than 60 kbp, the DNA coding region for genes with known function is shown in red. for homo sapiens and plasmodium-not much that codes
Structure of Mitochondrial Transporters
The structure of the ATP-ADP translocase is shown. • Notice that this structure comprises three similar units (shown in red, blue, and yellow) that come together to form a binding site, here occupied by atractyloside, an inhibitor of this transporter. • Other members of the mitochondrial transporter family adopt similar tripartite structures
Direct Observation of ATP-driven Rotation in ATP Synthase
The α3β3 hexamer of ATP synthase is fixed to a surface, with the γ subunit projecting upward and linked to a fluorescently labeled actin filament. • The addition and subsequent hydrolysis of ATP result in the counterclockwise rotation of the γ subunit, which can be directly seen under a fluorescence microscope.
Structure of Cytochrome c Oxidase (complex IV) ____ and _____ are embedded in the _____ ______ is the site of the reduction of oxygen to water the ______ is positioned near the intermembrane space to better accept electrons from _________ e- from cyt c to _________ to _______ to _________ to react with O2
This enzyme consists of 13 polypeptide chains. • Notice that most of the complex, as well as two major prosthetic groups (heme a and heme a3-CuB-close to each other) are embedded in the membrane (α helices represented by vertical tubes). • Heme a3-CuB is the site of the reduction of oxygen to water. • The CuA/CuA prosthetic group is positioned near the intermembrane space to better accept electrons from cytochrome c. e- from cyt c to CuA-CuA to heme A (in middle) to heme A3 the reaction with O2
Structure of Q-cytochrome c Oxidoreductase (brings e- from QH2 through complex III to cytochrome c)
This enzyme is a homodimer (two halves are identical) with each monomer consisting of 11 distinct polypeptide chains. Some of the more prominent components in one monomer are colored while the other monomer is white. -IRON SULFUR center with HIS • Although each monomer contains the same components, some are identified in one monomer or the other for ease of viewing. • Notice that the major prosthetic groups, three hemes (IRON) and a 2Fe-2S cluster (on edge to accept e- and bring them in), are located either near the cytoplasmic edge of the complex bordering the intermembrane space (top) or in the region embedded in the membrane (α helices represented by tubes). • They are well positioned to mediate the electrontransfer reactions between quinones in the membrane and cytochrome c in the intermembrane space
Mitochondrial Transporters
Transporters (also called carriers) are transmembrane proteins that carry specific ions and charged metabolites across the inner mitochondrial membrane. ATP-ADP carrier is ATP-ADP translocase dicarboxylate carrier: malate and phosphate back and forth carrier tricarboxylate carrier: citrate and H+ and back and forth with malate carrier phosphate carrier: OH- and phosphate carrier
Electron Flow from NADH to Molecular Oxygen Powers the Formation of a Proton Gradient (which is then used to make ATP)
We can calculate the ΔG°¢ for the reaction of NADH with O2. • Combining the two half-reactions and calculating ΔG°' .5O2 + 2H+ + 2e- -----H2O E0' = +0.82v (needs e-) NAD+ + H+ + 2e- ----NADH E0'= -0.32 V (donates e-) • ΔG°' can be calculated to have a value of −220.1 kJ mol−1 (−52.6 kcal mol−1). using the e- from NADH aad FADH2 from the O2 and use to power synthesis of ATP • Thus, the oxidation of one NADH can be coupled with the rephosphorylation of multiple ADP molecules.
Standard Reduction Potentials of Some Reactions
acceptors and donors chart -NAD+--NADH h=2; E0'= -0.32 will generate e- fairly small values
Measurement of Redox Potential
acid with conjugate base connected through agar bridge to acid with H2 gas -voltometer in between measuring e- flow
The High-potential Electrons of NADH Enter the Respiratory Chain at NADH-Q Oxidoreductase e- from NADH---_____---_____---_____---______ ___ protons pumped out of the mitochondrial matrix
e- from NADH--FMN--iron-sulfur clusters--Q--to QH2 The electrons from NADH are passed along to Q which is reduced to form QH2 by Complex I. -pumping out protons • QH2 leaves the enzyme for the Q pool in the hydrophobic interior of the inner mitochondrial membrane. • The electron carriers between NADH and Q include flavin mononucleotide (FMN) and several iron-sulfur clusters/proteins. • Four protons are simultaneously pumped out of the mitochondrial matrix by Complex I. (proton pump) NADH + Q (ubiquinone is reduced) + 5H+ from matrix ===== NAD+ + QH2 (ubiquinone) + 4H+ intermembrane space
Cytochrome c Oxidase (CIV) Mechanism
e- from cyt c--CuA-CuA compound on side--heme a--heme a3 (at center)--CuB (at center is Cu-accept e- reduced, bind O2) peroxide: a3-O-O-CuB + 4 protons and 2 e- 2 protons at a time from 2H2O to be released and left with original
Oxidation States of Quinones
fully oxidized-ubiquinone-the quinone we're talking about-oxidized form of coenzyme Q (Q ubiquinone): Q semiquinone intermediate (QH radical) one alcohol and one radical semiquinone radical ion (Q radical -) a radical and an O with 7 e- reduced form of coenzyme Q (QH2) fully reduced (from ketone to alcohol)
where do • The citric acid cycle and fatty acid oxidation occur?
• The citric acid cycle and fatty acid oxidation occur in the matrix.-- B-carbon bonds broken-degredation of fatty aids
