Scheffler Immo E. Mitochondria

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on control coefﬁ cients for the various enzymes as opposed to the simpler view

of rate limiting enzymes. How is the activity of the cycle adjusted to various

conditions in a given tissue such as muscle, and how is the cycle ’ s capacity

regulated in different tissues?

Before some answers to these questions can be proposed, a presentation of

the cycle and a limited discussion of the individual steps are in order. It is

logical to start with one reaction outside of the cycle that produces the impor-

tant intermediate acetyl - CoA. The overall reaction is

pyruvate CoA NAD NADH acetyl-CoA CO

++ → + +

The enzyme involved is a multienzyme complex named pyruvate dehydroge-

nase, consisting of 132 subunits (30 E

dimers, 60 E

dimers, and 6 E

dimers in

mammals) with a total molecular mass of several hundred thousand kilodaltons.

Five coenzymes are necessary: thiamine pyrophosphate, lipoamide, CoA, FAD,

and NAD. Substrates and intermediates are shuttled between the subunits,

facilitated by the highly organized, three - dimensional structure of this complex.

For a detailed discussion of the structure and mechanism the reader is referred

to standard biochemistry textbooks, or to the original literature.

Figure 6.1 presents an overview of the intermediates and their sequence in

the citric acid cycle. In this scheme the individual reactions have been num-

bered, and some brief comments about salient features of each reaction and

the corresponding enzyme will have to sufﬁ ce for the present treatment.

1. Citrate synthase catalyzes the condensation of acetyl - CoA with oxalo-

acetate to form citrate, a tricarboxylic acid from which a common name of the

cycle has been derived. The X - ray structure of the enzyme has been deter-

mined, and considerable detail about the mechanism is known. The reaction

can be described as a mixed aldol – Claisen condensation involving the stabili-

zation of the enol form of acetyl - CoA as an intermediate. Our current under-

standing of enzymes and their active sites also makes it clear why a speciﬁ c

chiral carbon is formed with the carboxymethyl group from acetyl - CoA con-

stituting the pro - S arm. A consequence of this stereochemistry is that the two

carbons entering the cycle from acetyl - CoA will not be liberated as CO

during

the ﬁ rst turn of the cycle.

2. Aconitase catalyzes the reversible interconversion between two struc-

tural isomers, citrate and isocitrate. Since citrate is prochiral, the two carboxy-

methyl groups of the symmetrical cityrate are distinguishable, and the hydroxyl

group is moved to the methylene carbon from oxaloacetate rather than to the

methylene group of the original acetyl - CoA. The enzyme is of special interest

because a nonheme iron – sulfur cluster ([4Fe – 4S]) participates in the active

site of the enzyme, although no electron transfer takes place. A speciﬁ c Fe(II)

in the cluster serves to bind and stabilize the substrates and intermediates.

More recently it was discovered that a cytosolic aconitase also serves as a

sensor of iron in the cytosol. Before its identity was established, this

THE KREBS CYCLE 301

302 METABOLIC PATHWAYS INSIDE MITOCHONDRIA

iron - responsive protein (IRP) had been shown to interact with iron - responsive

elements (IREs) on certain mRNAs. These IREs can be recognized as speciﬁ c

hairpin structures. When bound to IREs in the 5 ′ untranslated region of the

mRNAs for ferritin or 5 - aminolevulinate synthase, it prevents translation of

these mRNAs. Cytosolic aconitase binds to IREs when the iron concentration

is low; at high iron concentrations, aconitase is more tightly folded around the

[4Fe – 4S] cluster and incapable of binding the mRNA. An IRE is also found

in the 3 ′ untranslated region of the transferrin receptor mRNA. Binding of

aconitase (low iron) prevents turnover of the mRNA. At high iron concentra-

tions, degradation of the transferrin receptor mRNA is promoted. Thus, aconi-

tase as the iron sensor controls the transport of iron into the cell, the storage

of iron, a step in heme biosynthesis, and probably other pathways related to

iron (see references 1 – 3 for recent reviews).

3. Isocitrate dehydrogenase occurs in two forms in mammalian tissues. An

NAD - dependent form is found exclusively in mitochondria; a second form

Figure 6.1 The TCA (tri carboxylic acid) cycle or Krebs cycle.

acetyl - CoA

citrate

isocitrate

α-ketoglutarate

succinyl - CoA

succinate

fumarate

malate

oxaloacetate

NAD

NADH

NAD

NADH

FAD

FADH

NAD

NADH

COO

KREBS

CYCLE

COO

SCoA

COO

H-C-COO

COO

H-C-OH

C COO

utilizes NADP and is localized both in mitochondria and the cytosol. The reac-

tion produces the ﬁ rst of two CO

molecules produced per turn of the cycle,

and it links a decarboxylation to the oxidation of an alcohol to a keto group.

The ﬁ rst of three NADH molecules is also produced here.

4. The enzyme α - ketoglutarate dehydrogenase is a multifunctional enzyme

complex like the pyruvate dehydrogenase; and the third enzyme, dihydrolipoyl

dehydrogenase, is identical to that in PDH. The ﬁ rst two subunits are similar,

with speciﬁ city for the succinyl instead of acetyl moiety, and the ﬁ nal product

is another high - energy thioester, succinyl - CoA. NADH and CO

are the other

products of the reaction. At this point, two CO

molecules have been pro-

duced, accounting for the total input of two carbons from acetyl - CoA, but the

stereochemical considerations related to the formation of citrate and the sub-

sequent reactions can account for the observations that no radioactive CO

produced from [

C] acetyl - CoA during the ﬁ rst turn of the cycle.

5. Succinyl - CoA synthetase carries out a substrate - level phosphorylation

with the slight twist that GDP is the phosphate acceptor:

succinyl-CoA P GDP succinate GTP CoA

++ → + +

There is persuasive evidence from isotope tracer experiments that the

inorganic phosphate displaces the CoA to form succinyl - phosphate. The phos-

phate is transferred to GDP via a 3 - phospho - histidine intermediate on the

enzyme.

6. Succinate is oxidized to fumarate by the membrane - linked SDH complex.

A large (70 - kDa) ﬂ avoprotein forms the active site, and a covalently linked

ﬂ avin becomes the hydrogen acceptor to form FADH

. The ﬂ avin is linked to

a histidine side chain in a highly conserved portion of the protein. It is imme-

diately reoxidized with the release of protons and the transfer of two electrons

to the iron – sulfur centers of the iron – protein subunit of SDH. The Ip subunit

has three nonheme iron – sulfur centers ([2Fe – 2S], [3Fe – 4S], and [4Fe – 4S]) and

constitutes a prime example of a protein conducting electrons in the absence

of a heme group. The two integral membrane proteins of complex II are associ-

ated with a cytochrome, but it is still controversial whether this cytochrome

participates in the conduit of the electrons to the ubiquinone (CoQ). Succinate

dehydrogenase is the only Krebs cycle enzyme directly linked to the electron

transfer chain. A more detailed discussion of this complex can be found in

Chapter 5 .

7. Fumarate is hydrated by the addition of H

O to the double bond, cata-

lyzed by fumarase. Controversy about the mechanism has centered on the

question of whether the addition of OH

–

is the ﬁ rst step, leading to the forma-

tion of a carbanion that appears now to have been conﬁ rmed by

O exchange

experiments.

8. The ﬁ nal reaction catalyzed by malate dehydrogenase constitutes the last

oxidation step and hence production of another NADH.

THE KREBS CYCLE 303

304 METABOLIC PATHWAYS INSIDE MITOCHONDRIA

A ﬁ nal accounting of all the reactions with the inclusion of the pyruvate

dehydrogenase reactions leads to the following summation:

pyruvate P GDP NAD FAD 3 CO NADH FADH GTP

++ + + ⇒ + + +44

It should be reemphasized that pyruvate (C3) has become converted to three

molecules without any consumption of oxygen. If a steady supply of

NAD

was available, glucose could thus be broken down to carbon dioxide in

the absence of oxygen. The cost of NAD

makes this impossible, and in reality

it has to be obtained by recycling. Hence, oxidation of NADH by complex I

of the electron transfer chain is essential to keep the citric acid cycle going.

Accumulation of NADH causes a strong inhibition of pyruvate dehydroge-

nase and α - ketoglutarate dehydrogenase, and the author ’ s laboratory has iso-

lated mammalian cell mutants with defects in the electron transfer chain based

on the almost total inhibition of the Krebs cycle (4) . Substrate and product

concentrations are the immediate controlling factors determining the ﬂ ux

through the cycle, and ATP, ADP, and Ca

are allosteric effectors of citric acid

cycle enzymes. Overall, oxygen consumption, NADH oxidation, and ATP syn-

thesis are tightly coupled not only to each other, but also to the citric acid

cycle by the above mechanisms.

In the end, Lavoisier was correct in viewing respiration as combustion, but

little did he suspect that many steps are necessary to make this “ burning ” of

glucose in a living cell such a controlled process. The many steps involved not

only ensure control over a “ slow burn, ” but also permit the efﬁ cient capture

of the free energy released in the process, along with its interconversion and

utilization in many biological processes. It all started with attempts to under-

stand respiration, followed by a curiosity about the biochemistry and ener-

getics of muscular contraction. As a ﬁ nal thought, it may be worthwhile to be

made aware that an average person turns over more than his/her own body-

weight of ATP per day, depending on the level of activity.

6.3 FATTY ACID METABOLISM

Fatty acid oxidation was one of the ﬁ rst metabolic pathways ﬁ rmly localized

in mitochondria by the pioneering studies of Lehninger and Kennedy in the

late 1940s. Since fats and fatty acids derived from them are obvious energy

sources, their degradation in mitochondria must have been suggestive. Soon

thereafter the localization of the Krebs cycle enzymes in the same organelle

allowed the integration of fatty acid oxidation with the TCA cycle via the

important intermediate acetyl - CoA. Peroxisomes had not been recognized at

that time, and therefore the discovery of β - oxidation of fatty acids in peroxi-

somes did not confuse the issue. It is now known that peroxisomes in animal

cells “ specialize ” in the degradation of very long fatty acids ( > 22 carbon

atoms), and they may even just shorten them to be accepted by the mitochon-

drial system. In plant cells, however, fatty acid degradation is restricted to

peroxisomes and glyoxysomes. The latter are specialized peroxisomes with

special signiﬁ cance in metabolism during seed germination.

Before considering the details, it will be instructive to take a more global

view. The fatty acids are ﬁ rst made available in the cytosol — for example,

from the hydrolysis of cholesterol esters of low - density lipoprotein (LDL) in

lysosomes or from the degradation of phospholipids by lipases. Their trans-

port into mitochondria is not a trivial issue (see below). The ﬁ nal product of

their degradation is acetyl - CoA that can feed into the Krebs cycle. Under

different conditions, fatty acids can also be synthesized, also starting from

acetyl - CoA. The biosynthesis of fatty acids does not occur simply by the

reversal of the reactions of the β - oxidation, an observation that generally

applies to other pathways such as glycolysis and gluconeogenesis. Different

enzymes are utilized; and most signiﬁ cantly, the biosynthesis of fatty acids

takes place in the cytosol. During elongation in the cytosol the acyl group is

attached to a large multifunctional protein (in animal cells) taking the place

of CoA.

Fatty acid degradation generates acetyl - CoA as the ﬁ nal product, but inter-

mediates are also attached to CoA during the entire process. Thus, the ﬁ rst

step in fatty acid degradation requires an activation:

Fatty acid CoA ATP acyl-CoA AMP PP

++→ + +

Several acyl - CoA - sythetases (thiokinases) serve in this activation in the

cytosol. To reach the mitochondrial matrix, a shuttle system has evolved, which

is shown in Figure 6.2 . The acyl group is transferred to carnitine, liberating

CoA. Acyl - carnitine is transported into the mitochondria by a speciﬁ c carrier

protein; and once it is inside, the transesteriﬁ cation is reversed to form carni-

tine and acyl - CoA. The carrier protein is operating like an antiporter, trans-

porting free carnitine out of the mitochondria.

The β - oxidation reactions (Figure 6.3 ) are well known and found in all

standard biochemistry textbooks. A brief listing of all reactions and some

select comments will sufﬁ ce here.

The ﬁ rst reaction is an an elimination of hydrogen to form a double bond

between the α and β carbons of acyl - CoA.

acyl-CoA FAD - 2-enoyl-CoA FADH

+⇒ +trans Δ

(1)

The FAD is a cofactor of acyl - CoA dehydrogenase, and the FADH

is reoxi-

dized via two proteins, the electron transfer ﬂ avoprotein (ETF) and the ETF:

ubiquinone oxidoreductase transferring electrons to ubiquinone (to yield

), which is reoxidized via the electron transfer chain. Superﬁ cially, the

acyl - CoA dehydrogenase therefore resembles succinate dehydrogenase in the

use of ﬂ avin as the immediate acceptor of hydrogen, followed by electron

transfers via iron – sulfur centers to ubiquinone.

trans- 2-enoyl-CoA H O 3- hydroxyacyl-CoA

L-Δ+⇒

(2)

FATTY ACID METABOLISM 305

306 METABOLIC PATHWAYS INSIDE MITOCHONDRIA

This hydration reaction resembles to formation of malate from fumarate and

is catalyzed by enoyl - CoA hydratase. It is followed by an oxidation to

β - ketoacyl - CoA:

3- hydroxyacyl-CoA NAD -ketoacyl-CoA NADHL-

+⇒ +β (3)

In the ﬁ nal step, another CoA is a second substrate, and the reaction splits off

a two - carbon unit to form acetyl - CoA, leaving acyl - CoA, but now two carbon

atoms shorter than at the start:

() ( )C acyl-CoA CoA C acyl-CoA acetyl-CoA

+⇒ +

−2

(4)

Figure 6.2 Acyl - CoA/carnitine shuttle in mitochondria.

CYTOSOL

MITOCHONDRIA

carnitine

acyl-CoA

acyl-carnitine

SCoAR

COO

CHCH

N(CH )

COO

CHCH

N(CH )

H-SCoA

acyl-CoA

H-SCoA

carnitine

acyl-carnitine

carnitine

acyl-carnitine

acyl-CoA

H-SCoA

Figure 6.3 β - oxidation reactions.

acyl-CoA dehydrogenase

enoyl-CoA-hydratase

3-L-hydroxyacyl-CoA-dehydrogenase

β-ketoacyl-CoA-thiolase

SCoA

CH (CH )

SCoA

CH (CH )

SCoA

CH (CH )

SCoA

CH (CH )

SCoA

CH (CH )

FATTY ACID METABOLISM 307

308 METABOLIC PATHWAYS INSIDE MITOCHONDRIA

The series of reactions 1 – 4 is repeated, and a saturated fatty acid with an

even number of carbons can be converted to acetyl - CoA with no further

complications, except that different acyl - CoA dehydrogenases are used as

the carbon chain of the acyl group gets shorter. Unsaturated fatty acids

can be dealt with by a combination of isomerases and reductases (requiring

NADPH), depending on the position of the double bonds and their con-

ﬁ guration. The β - oxidation of odd - chain fatty acids leads to the formation

of propionyl - CoA, which must be dealt with by a distinct series of

reactions:

(1)

propionyl-CoA ATP CO -methylmalonyl-CoA ADP P

++→ + +()S

(2)

( -methylmalonyl-CoA -methylmalonyl-CoASR)()→

(3)

()R -methylmalonyl-CoA succinyl-CoA→

Reactions 1 – 3 are catalyzed by a carboxylase, a racemase and a mutase,

respectively. The methyl - malonyl - CoA mutase is an unusual enzyme requiring

5 - deoxyadenosylcobalamin (coenzyme B

) as a cofactor; this cofactor con-

tains Co(III) in the center of a ring resembling heme and has the only carbon –

metal bond known in biology. Vitamin B

deﬁ ciency was ﬁ rst shown in 1926

to cause pernicious anemia, and humans must acquire it from their food

(meat). Uptake and transport in the bloodstream requires speciﬁ c proteins,

cobalamins. The solution of the crystal structure of cobalamin by D. Hodgkin

was a notable achievement in crystallography in 1956, which was later

dwarfed only by the determination of the crystal structure of whole proteins.

Cobinamide, a precursor in cobalamin biosynthesis, has an extremely high

afﬁ nity for cyanide, and it has been proposed to be used in cyanide detoxiﬁ ca-

tion (5) .

While the acetyl - CoA formed during β - oxidation could enter the Krebs

cycle as expected, its actual fate in liver may be different. A process referred

to as ketogenesis converts acetyl - CoA into compounds called ketone bodies,

which include acetone, acetoacetate, and β - hydroxybutyrate. The solubility of

ketone bodies makes them readily transportable by the blood to other organs

and tissues where they serve as alternate metabolic fuels. For example, when

fats are mobilized during starvation, ketone bodies can substitute for glucose

in the brain.

Although the biosynthesis of fatty acids takes place in the cytosol, and its

details get short shrift in this book on mitochondria, it is appropriate and rel-

evant to discuss one of the ﬁ rst steps required for this cyctosolic sequence to

take place: Acetyl - CoA has to get out of the mitochondria. This is achieved

by making citrate in the mitochondria, which can be exported by a transport

system for tribarboxylic acids. In the cytosol, acetyl - CoA is regenerated by

ATP - citrate lyase:

citrate ATP CoA acetyl-CoA ADP P oxaloacetate

++⇒ + ++

6.4 THE UREA CYCLE

The Krebs cycle ketoacids α - ketoglutarate and oxaloacetate can be withdrawn

to form amino acids by transamination or added to the cycle by the easily

reversible reaction, and it may be worth mentioning that mammalian cells

in tissue culture thrive in media with added glutamine even though it could

be considered a nonessential amino acid. It enters the Krebs cycle as a -

ketoglutarate, thus providing nitrogen in a transamination reaction. This

nitrogen is recaptured when oxaloacetate is converted to aspartate (and aspar-

agine). In ﬁ broblasts these two nonessential amino acids are derived almost

exclusively from glutamine via the operation of the Krebs cycle (4, 6) .

A most signiﬁ cant role of mitochondria in amino acid metabolism is the par-

ticipation in the urea cycle. The urea cycle was the ﬁ rst metabolic cycle discov-

ered and described by Krebs ﬁ ve years before his more famous citric acid cycle.

The cycle is crucial for the breakdown of excess amino acids and the conversion

of excess nitrogen into urea, the excretable form of nitrogen in many terrestial

animals. The major activity takes place in the liver, from where urea is deliverd

via the bloodstream to the kidneys for excretion in urine.

As expected, there is one reaction creating a precursor, carbamoyl -

phosphate, which can enter the cycle by condensation with a cycle interme-

diate, and products including urea are generated in various steps in the cycle.

Only some reactions of the cycle take place in mitochondria; it is completed

in the cytosol. The complete cycle is shown in Figure 6.4 . The two reactions

taking place in mitochondria are the formation of carbamoyl phosphate from

ammonia and bicarbonate, driven by the hydrolysis of ATP, and the condensa-

tion of carbamoyl phosphate with ornithine to form citrulline. Carbamoyl

phosphate synthetase I, the enzyme required for step 1, is allosterically regu-

lated by N - acetylglutamate, made from glutamate and acetyl - CoA. Increased

glutamate concentrations signal excess amino acids (nitrogen), and an increase

in N - acetylglutamate activates carbamoyl phosphate synthesis.

6.5 BIOSYNTHESIS OF HEME

Studies on respiration, cytochromes, and heme leading to the elucidation of

the role of mitochondria have been intricately linked for over a century. In

hemoglobin in the blood, the heme group is used to bind and transport oxygen.

In the electron transfer chain, heme groups associated with peptides form the

cytochromes. Another important cytochrome is the cytochrome P

450

, an enzyme

system used primarily in the liver for metabolizing various toxic compounds

such as carcinogens. An intermediate in heme biosynthesis is a tetrapyrrole

derivative (see below), a conjugated, planar ring structure without the metal

ion, and in plants this intermediate is the precursor of chlorophyll.

The use of isotope tracers made it possible in the late 1940s to investigate

the precursors for heme biosynthesis, and the whole pathway was established

BIOSYNTHESIS OF HEME 309

310 METABOLIC PATHWAYS INSIDE MITOCHONDRIA

in the following decade (Figure 6.5 ). It starts in the mitochondria with a Krebs

cycle intermediate, succinyl - CoA, and the formation of δ - aminolevulinic acid

(ALA), involving the rate - limiting enzyme ALA synthase. It was described

earlier how ALA protein levels can be controlled by regulating the efﬁ ciency

with which ALA mRNA is translated (see also below). ALA leaves the mito-

chondria to be converted to coproporphyrinogen III in a complex series of

reactions. An oxidative modiﬁ cation of the ring structure is coupled to its

transport back into mitochondria, where the synthesis of heme is completed

with another oxidation and the ﬁ nal addition of the iron to the center of the

porphyrin ring. It it noteworthy that the latter reactions require molecular

oxygen and thus are sensitive to oxygen concentrations.

ALA synthase is the key regulatory enzyme in the liver, but may not play

this role in immature erythrocytes, another tissue in which heme biosynthesis

is prominent. Under these conditions, ALA synthase may be feedback inhib-

ited by its product. Its import into mitochondria may be controlled. It is also

Figure 6.4 Urea cycle.

COO

(CH

)

COO

carbamoyl phosphate

citrulline

arginino-

succinate

fumarate

arginine

ornithine

urea

ornithine

citrulline

aspartate

ATP

AMP + PP

MITOCHONDRION

UREA

CYCLE

CYTOSOL

COO

(CH

)

COO

(CH

)

(CH

)

HC NH

COO

2ATP + HCO + NH

2ADP + P