Berg J.M., Tymoczko J.L., Stryer L. Biochemistry

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(a) What is the effect of the absence of isocitrate lyase?

The techniques discussed in Chapter 6 were used to reinsert the gene encoding isocitrate lyase into bacteria from

which it had previously been deleted.

In graph B, black circles represent bacteria into which the gene was reinserted and red circles bacteria from which

the gene was still missing.

[Data after McKinney et al., 2000. Nature 406:735

738.]

(b) Do these results support those obtained in part a?

(d) Why do these bacteria perish in the absence of the glyoxylate cycle?

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See answer

II. Transducing and Storing Energy 17. The Citric Acid Cycle Problems

Effect of citrate on oxygen consumption by minced pigeon breast muscle

II. Transducing and Storing Energy 17. The Citric Acid Cycle Problems

Disappearance of citric acid in pigeon breast muscle in the presence of arsenite

II. Transducing and Storing Energy 17. The Citric Acid Cycle

Selected Readings

Where to start

L.J. Reed and M.L. Hackert. 1990. Structure-function relationships in dihydrolipoamide acyltransferases J. Biol. Chem.

265: 8971-8974. (PubMed)

A. Mattevi, G. Obmolova, E. Schulze, K.H. Kalk, A.H. Westphal, A. De Kok, and W.G. Hol. 1992. Atomic structure of

the cubic core of the pyruvate dehydrogenase multienzyme complex Science 255: 1544-1550. (PubMed)

Pyruvate dehydrogenase complex

T. Izard, A. Ævarsson, M.D. Allen, A.H. Westphal, R.N. Perham, A. De Kok, and W.G. Hol. 1999. Principles of quasi-

equivalence and Euclidean geometry govern the assembly of cubic and dodecahedral cores of pyruvate dehydrogenase

complexes Proc. Natl. Acad. Sci. USA 96: 1240-1245. (PubMed) (Full Text in PMC)

A. Ævarsson, K. Seger, S. Turley, J.R. Sokatch, and W.M.J. Hol. 1999. Crystal structure of 2-oxoisovalerate and

dehydrogenase and the architecture of 2-oxo acid dehydrogenase multiple enzyme complexes Nat. Struct. Biol. 6: 785-

792. (PubMed)

G.J. Domingo, H.J. Chauhan, I.A. Lessard, C. Fuller, and R.N. Perham. 1999. Self-assembly and catalytic activity of the

pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus Eur. J. Biochem. 266: 1136-1146.

(PubMed)

D.D. Jones, H.J. Horne, P.A. Reche, and R.N. Perham. 2000. Structural determinants of post-translational modification

and catalytic specificity for the lipoyl domains of the pyruvate dehydrogenase multienzyme complex of Escherichia coli

J. Mol. Biol. 295: 289-306. (PubMed)

R.G. McCartney, J.E. Rice, S.J. Sanderson, V. Bunik, H. Lindsay, and J.G. Lindsay. 1998. Subunit interactions in the

mammalian alpha-ketoglutarate dehydrogenase complex: Evidence for direct association of the alpha-ketoglutarate

dehydrogenase and dihydrolipoamide dehydrogenase components J. Biol. Chem. 273: 24158-24164. (PubMed)

Structure of citric acid cycle enzymes

A.D. Chapman, A. Cortes, T.R. Dafforn, A.R. Clarke, and R.L. Brady. 1999. Structural basis of substrate specificity in

malate dehydrogenases: Crystal structure of a ternary complex of porcine cytoplasmic malate dehydrogenase, alpha-

ketomalonate and tetrahydoNAD J. Mol. Biol. 285: 703-712. (PubMed)

Fraser, M. E., James, M. N., Bridger, W. A., and Wolodko, W. T., 1999. A detailed structural description of Escherichia

coli succinyl-CoA synthetase. J. Mol. Biol. 285:1633 1653. [Published erratum appears in May 7, 1999, issue of J.

Mol. Biol. 288(3):501.]

S.J. Lloyd, H. Lauble, G.S. Prasad, and C.D. Stout. 1999. The mechanism of aconitase: 1.8 Å resolution crystal structure

of the S642a:citrate complex. Protein Sci. 8: 2655-2662. (PubMed)

S.J. Remington. 1992. Structure and mechanism of citrate synthase Curr. Top. Cell. Regul. 33: 209-229. (PubMed)

I.A. Rose. 1998. How fumarase recycles after the malate fumarate reaction: Insights into the reaction mechanism

Biochemistry 37: 17651-17658. (PubMed)

J.D. Johnson, W.W. Muhonen, and D.O. Lambeth. 1998. Characterization of the ATP- and GTP-specific succinyl-CoA

synthetases in pigeon: The enzymes incorporate the same subunit J. Biol. Chem. 273: 27573-27579. (PubMed)

M. Karpusas, B. Branchaud, and S.J. Remington. 1990. Proposed mechanism for the condensation reaction of citrate

synthase: 1.9-Å structure of the ternary complex with oxaloacetate and carboxymethyl coenzyme A. Biochemistry 29:

2213-2219. (PubMed)

H. Lauble, M.C. Kennedy, H. Beinert, and C.D. Stout. 1992. Crystal structures of aconitase with isocitrate and

nitroisocitrate bound Biochemistry 31: 2735-2748. (PubMed)

Organization of the citric acid cycle

C. Velot, M.B. Mixon, M. Teige, and P.A. Srere. 1997. Model of a quinary structure between Krebs TCA cycle

enzymes: A model for the metabolon Biochemistry 36: 14271-14276. (PubMed)

S.J. Barnes and P.D. Weitzman. 1986. Organization of citric acid cycle enzymes into a multienzyme cluster FEBS Lett.

201: 267-270. (PubMed)

P.M. Haggie and K.M. Brindle. 1999. Mitochondrial citrate synthase is immobilized in vivo J. Biol. Chem. 274: 3941-

3945. (PubMed)

I. Morgunov and P.A. Srere. 1998. Interaction between citrate synthase and malate dehydrogenase: Substrate channeling

of oxaloacetate J. Biol. Chem. 273: 29540-29544. (PubMed)

Regulation

B. Huang, R. Gudi, P. Wu, R.A. Harris, J. Hamilton, and K.M. Popov. 1998. Isoenzymes of pyruvate dehydrogenase

phosphatase: DNA-derived amino acid sequences, expression, and regulation J. Biol. Chem. 273: 17680-17688.

(PubMed)

M. Bowker-Kinley and K.M. Popov. 1999. Evidence that pyruvate dehydrogenase kinase belongs to the ATPase/kinase

superfamily Biochem. J. 1: 47-53.

S. Jitrapakdee and J.C. Wallace. 1999. Structure, function and regulation of pyruvate carboxylase Biochem. J. 340: 1-16.

(PubMed)

J.H. Hurley, A.M. Dean, J.L. Sohl, D.J. Koshland, and R.M. Stroud. 1990. Regulation of an enzyme by phosphorylation

at the active site Science 249: 1012-1016. (PubMed)

Evolutionary aspects

E. Meléndez-Hevia, T.G. Waddell, and M. Cascante. 1996. The puzzle of the Krebs citric acid cycle: Assembling the

pieces of chemically feasible reactions, and opportunism in the design of metabolic pathways in evolution J. Mol. Evol.

43: 293-303. (PubMed)

J.E. Baldwin and H. Krebs. 1981. The evolution of metabolic cycles Nature 291: 381-382. (PubMed)

H. Gest. 1987. Evolutionary roots of the citric acid cycle in prokaryotes Biochem. Soc. Symp. 54: 3-16. (PubMed)

P.D.J. Weitzman. 1981. Unity and diversity in some bacterial citric acid cycle enzymes Adv. Microbiol. Physiol. 22: 185-

244.

Discovery of the citric acid cycle

H.A. Krebs and W.A. Johnson. 1937. The role of citric acid in intermediate metabolism in animal tissues Enzymologia 4:

148-156.

H.A. Krebs. 1970. The history of the tricarboxylic acid cycle Perspect. Biol. Med. 14: 154-170. (PubMed)

Krebs, H. A., and Martin, A., 1981. Reminiscences and Reflections. Clarendon Press.

II. Transducing and Storing Energy

18. Oxidative Phosphorylation

The NADH and FADH

formed in glycolysis, fatty acid oxidation, and the citric acid cycle are energy-rich molecules

because each contains a pair of electrons having a high transfer potential. When these electrons are used to reduce

molecular oxygen to water, a large amount of free energy is liberated, which can be used to generate ATP. Oxidative

phosphorylation is the process in which ATP is formed as a result of the transfer of electrons from NADH or FADH

by a series of electron carriers. This process, which takes place in mitochondria, is the major source of ATP in

aerobic organisms (Figure 18.1). For example, oxidative phosphorylation generates 26 of the 30 molecules of ATP that

are formed when glucose is completely oxidized to CO

and H

Oxidative phosphorylation is conceptually simple and mechanistically complex. Indeed, the unraveling of the

mechanism of oxidative phosphorylation has been one of the most challenging problems of biochemistry. The flow of

electrons from NADH or FADH

to O

through protein complexes located in the mitochondrial inner membrane leads to

the pumping of protons out of the mitochondrial matrix. The resulting uneven distribution of protons generates a pH

gradient and a transmembrane electrical potential that creates a proton-motive force. ATP is synthesized when protons

flow back to the mitochondrial matrix through an enzyme complex. Thus, the oxidation of fuels and the phosphorylation

of ADP are coupled by a proton gradient across the inner mitochondrial membrane (Figure 18.2).

Oxidative phosphorylation is the culmination of a series of energy transformations that are called cellular respiration or

simply respiration in their entirety. First, carbon fuels are oxidized in the citric acid cycle to yield electrons with high

transfer potential. Then, this electron-motive force is converted into a proton-motive force and, finally, the proton-motive

force is converted into phosphoryl transfer potential. The conversion of electron-motive force into proton-motive force is

carried out by three electron-driven proton pumps

NADH-Q oxidoreductase, Q-cytochrome c oxidoreductase, and

cytochrome c oxidase. These large transmembrane complexes contain multiple oxidation-reduction centers, including

quinones, flavins, iron-sulfur clusters, hemes, and copper ions. The final phase of oxidative phosphorylation is carried

out by ATP synthase, an ATP-synthesizing assembly that is driven by the flow of protons back into the mitochondrial

matrix. Components of this remarkable enzyme rotate as part of its catalytic mechanism. Oxidative phosphorylation

vividly shows that proton gradients are an interconvertible currency of free energy in biological systems.

Respiration

An ATP-generating process in which an inorganic compound (such

as molecular oxygen) serves as the ultimate electron acceptor. The

electron donor can be either an organic compound or an inorganic

one.

II. Transducing and Storing Energy 18. Oxidative Phosphorylation

Figure 18.1. Electron Micrograph of a Mitochondrion. [Courtesy of Dr. George Palade.]

II. Transducing and Storing Energy 18. Oxidative Phosphorylation

Figure 18.2. Essence of Oxidative Phosphorylation. Oxidation and ATP synthesis are coupled by transmembrane

proton fluxes.

II. Transducing and Storing Energy 18. Oxidative Phosphorylation

Mitochondria, Stained Green, Form a Network Inside a Fibroblast Cell (Left). Mitochondria oxidize carbon fuels to

form cellular energy. This transformation requires electron transfer through several large protein complexes (above),

some of which pump protons, forming a proton gradient that powers the synthesis of ATP. [(Left) Courtesy of Michael

P. Yaffee, Dept. of Biology, University of California at San Diego.]

II. Transducing and Storing Energy 18. Oxidative Phosphorylation

18.1. Oxidative Phosphorylation in Eukaryotes Takes Place in Mitochondria

Mitochondria are oval-shaped organelles, typically about 2 µm in length and 0.5 µm in diameter, about the size of a

bacterium. Eugene Kennedy and Albert Lehninger discovered a half-century ago that mitochondria contain the

respiratory assembly, the enzymes of the citric acid cycle, and the enzymes of fatty acid oxidation.

18.1.1. Mitochondria Are Bounded by a Double Membrane

Electron microscopic studies by George Palade and Fritjof Sjöstrand revealed that mitochondria have two membrane

systems: an outer membrane and an extensive, highly folded inner membrane. The inner membrane is folded into a

series of internal ridges called cristae. Hence, there are two compartments in mitochondria: (1) the intermembrane space

between the outer and the inner membranes and (2) the matrix, which is bounded by the inner membrane (Figure 18.3).

Oxidative phosphorylation takes place in the inner mitochondrial membrane, in contrast with most of the reactions of the

citric acid cycle and fatty acid oxidation, which take place in the matrix.

The outer membrane is quite permeable to most small molecules and ions because it contains many copies of

mitochondrial porin, a 30

35 kd poreforming protein also known as VDAC, for voltage-dependent anion channel.

VDAC plays a role in the regulated flux of metabolites usually anionic species such as phosphate, chloride, organic

anions, and the adenine nucleotides across the outer membrane. VDAC appears to form an open β -barrel structure

similar to that of the bacterial porins (Section 12.5.2), although mitochondrial porins and bacterial porins may have

evolved independently. Some cytoplasmic kinases bind to VDAC, thereby obtaining preferential access to the exported

ATP. In contrast, the inner membrane is intrinsically impermeable to nearly all ions and polar molecules. A large family

of transporters shuttles metabolites such as ATP, pyruvate, and citrate across the inner mitochondrial membrane. The

two faces of this membrane will be referred to as the matrix side and the cytosolic side (the latter because it is freely

accessible to most small molecules in the cytosol). They are also called the N and P sides, respectively, because the

membrane potential is negative on the matrix side and positive on the cytosolic side.

In prokaryotes, the electron-driven proton pumps and ATP-synthesizing complex are located in the cytoplasmic

membrane, the inner of two membranes. The outer membrane of bacteria, like that of mitochondria, is permeable to most

small metabolites because of the presence of porins.

18.1.2. Mitochondria Are the Result of an Endosymbiotic Event

Mitochondria are semiautonomous organelles that live in an endosymbiotic relation with the host cell. These

organelles contain their own DNA, which encodes a variety of different proteins and RNAs. The genomes of

mitochondrial range broadly in size across species. The mitochondrial genome of the protist Plasmodium falciparum

consists of fewer than 6000 base pairs (6 kbp), whereas those of some land plants comprise more than 200 kbp (Figure

18.4). Human mitochondrial DNA comprises 16,569 bp and encodes 13 respiratory-chain proteins as well as the small

and large ribosomal RNAs and enough tRNAs to translate all codons. However, mitochondria also contain many

proteins encoded by nuclear DNA. Cells that contain mitochondria depend on these organelles for oxidative

phosphorylation, and the mitochondria in turn depend on the cell for their very existence. How did this intimate

symbiotic relation come to exist?

An endosymbiotic event is thought to have occurred whereby a freeliving organism capable of oxidative phosphorylation

was engulfed by another cell. The double membrane, circular DNA (with some exceptions), and mitochondrial-specific

transcription and translation machinery all point to this conclusion. Thanks to the rapid accumulation of sequence data

for mitochondrial and bacterial genomes, it is now possible to speculate on the origin of the "original" mitochondrion

with some authority. The most mitochondrial-like bacterial genome is that of Rickettsia prowazekii, the cause of louse-

borne typhus. The genome for this organism is more than 1 million base pairs in size and contains 834 protein-encoding

genes. Sequence data suggest that all extant mitochondria are derived from an ancestor of R. prowazekii as the result of a

single endosymbiotic event.

The evidence that modern mitochondria result from a single event comes from examination of the most bacteria-like

mitochondrial genome, that of the protozoan Reclinomonas americana. Its genome contains 97 genes, of which 62

specify proteins that include all of the protein-coding genes found in all of the sequenced mitochondrial genomes (Figure

18.5). Yet, this genome encodes less than 2% of the protein-coding genes in the bacterium E. coli. It seems unlikely that

mitochondrial genomes resulting from several endosymbiotic events could have been independently reduced to the same

set of genes found in R. americana.

Note that transient engulfment of prokaryotic cells by larger cells is not uncommon in the microbial world. In regard to

mitochondria, such a transient relation became permanent as the bacterial cell lost DNA, making it incapable of

independent living, and the host cell became dependent on the ATP generated by its tenant.

II. Transducing and Storing Energy 18. Oxidative Phosphorylation 18.1. Oxidative Phosphorylation in Eukaryotes Takes Place in Mitochondria

Publishing Company, Inc., Belmost, California 94002. Adapted by permission of the publisher.]

II. Transducing and Storing Energy 18. Oxidative Phosphorylation 18.1. Oxidative Phosphorylation in Eukaryotes Takes Place in Mitochondria

Figure 18.4. Sizes of Mitochondrial Genomes. The sizes of three mitochondrial genomes compared with the genome of

Rickettsia, a relative of the 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.

II. Transducing and Storing Energy 18. Oxidative Phosphorylation 18.1. Oxidative Phosphorylation in Eukaryotes Takes Place in Mitochondria

Figure 18.5. Overlapping Gene Complements of Mitochondria. 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. [After M. W.

Gray, G. Burger, and B. F. Lang. Science 283(1999): 1476

1481.]

II. Transducing and Storing Energy 18. Oxidative Phosphorylation

18.2. Oxidative Phosphorylation Depends on Electron Transfer

In Chapter 17, the primary function of the citric acid cycle was identified as the generation of NADH and FADH

by the

oxidation of acetyl CoA. In oxidative phosphorylation, NADH and FADH

are used to reduce molecular oxygen to

water. The highly exergonic reduction of molecular oxygen by NADH and FADH

occurs in a number of electron-

transfer reactions, taking place in a set of membrane proteins known as the electron-transport chain.

18.2.1. High-Energy Electrons: Redox Potentials and Free-Energy Changes

High-energy electrons and redox potentials are of fundamental importance in oxidative phosphorylation. In oxidative

phosphorylation, the electron transfer potential of NADH or FADH

is converted into the phosphoryl transfer potential

of ATP. We need quantitative expressions for these forms of free energy. The measure of phosphoryl transfer potential is

already familiar to us: it is given by ∆ G°´ for the hydrolysis of the activated phosphate compound. The corresponding

expression for the electron transfer potential is E´

, the reduction potential (also called the redox potential or oxidation-

reduction potential).

The reduction potential is an electrochemical concept. Consider a substance that can exist in an oxidized form X and a

reduced form X

. Such a pair is called a redox couple. The reduction potential of this couple can be determined by

measuring the electromotive force generated by a sample half-cell connected to a standard reference half-cell (Figure

18.6). The sample half-cell consists of an electrode immersed in a solution of 1 M oxidant (X) and 1 M reductant (X

The standard reference half-cell consists of an electrode immersed in a 1 M H

solution that is in equilibrium with H

gas at 1 atmosphere pressure. The electrodes are connected to a voltmeter, and an agar bridge establishes electrical

continuity between the half-cells. Electrons then flow from one half-cell to the other. If the reaction proceeds in the

direction

the reactions in the half-cells (referred to as half-reactions or couples) must be

Thus, electrons flow from the sample half-cell to the standard reference half-cell, and the sample-cell electrode is taken

to be negative with respect to the standard-cell electrode. The reduction potential of the X:X

couple is the observed

voltage at the start of the experiment (when X, X

, and H

are 1 M). The reduction potential of the H

couple is

defined to be 0 volts.

The meaning of the reduction potential is now evident. A negative reduction potential means that the reduced form of a

substance has lower affinity for electrons than does H

, as in the preceding example. A positive reduction potential

means that the reduced form of a substance has higher affinity for electrons than does H

. These comparisons refer to

standard conditions

namely, 1 M oxidant, 1 M reductant, 1 M H

, and 1 atmosphere H

. Thus, a strong reducing agent

(such as NADH) is poised to donate electrons and has a negative reduction potential, whereas a strong oxidizing agent

(such as O

) is ready to accept electrons and has a positive reduction potential.

The reduction potentials of many biologically important redox couples are known (Table 18.1). Table 18.1 is like those

presented in chemistry texts except that a hydrogen ion concentration of 10

-7

M (pH 7) instead of 1 M (pH 0) is the

standard state adopted by biochemists. This difference is denoted by the prime in E´

. Recall that the prime in ∆ G°´

denotes a standard free-energy change at pH 7.

The standard free-energy change ∆ G°´ is related to the change in reduction potential ∆ E´

in which n is the number of electrons transferred, F is a proportionality constant called the faraday [23.06 kcal mol

-1

(96.48 kJ mol

-1

)], ∆ E´

is in volts, and ∆ G°´ is in kilocalories or kilojoules per mole.

The free-energy change of an oxidation-reduction reaction can be readily calculated from the reduction potentials of the

reactants. For example, consider the reduction of pyruvate by NADH, catalyzed by lactate dehydrogenase.

The reduction potential of the NAD

:NADH couple, or half-reaction, is -0.32 V, whereas that of the pyruvate: lactate

couple is -0.19 V. By convention, reduction potentials (as in Table 18.1) refer to partial reactions written as reductions:

oxidant + e

reductant. Hence,

To obtain reaction a from reactions b and c, we need to reverse the direction of reaction c so that NADH appears on the

left side of the arrow. In doing so, the sign of E´

must be changed.