Biología de los Microorganismos
46
Transcript of Biología de los Microorganismos
Capítulo 1: Estructura y metabolismo celularContenidos
1.1 Microorganismos y microbiología
1.2 Estructura y funciones de la célula
1.3 Metabolismo microbiano y bioquímica básica
1.4 Diversidad metabólica en los microorganismos
1.5 Calidad microbiológica del agua y su potabilizaciónTratamiento de aguas residuales
1.3 Metabolismo microbiano y bioquímica básicaMetabolismo microbiano
Metabolismo: suma de las transformaciones químicas que ocurren en la célula.
Metabolismo microbianoFunciones del metabolismo
Obtener energía
química del entorno
Almacenarla
Utilizarla en diferentes funciones celulares
Nutrientes exógenos (azúcares)
Convertirlos en unidades precursoras
(monómeros)
Convertirlos a macromolé-
culas
Formar y degradar moléculas
Para funciones
específicas
Ej. Movilidad y captación
de nutrientes
a) Nutrición microbianaü Los nutrientes son sustancias que se emplean en la biosíntesis y producción de energía.
ü Son necesarios para el crecimiento microbiano.
ü Los nutrientes aportan a las células los ingredientes químicos que necesitan para la síntesis de monómeros, que son los componentes de las macromoléculas, que a su vez construyen estructuras celulares.
Monómeros Macromoléculas Estructuras celularesMonosacáridos Polisacáridos Pared celular
GlicocálixAminoácidos Proteínas Membrana
RibosomasFlageloPelos y fimbriasEnzimas
Ácidos grasos Lípidos Membranas
Bases nitrogenadas Ácidos nucleicos Cromosoma RibosomasPlásmidos
Metabolismo microbiano a) Nutrición microbiana
b) Medio de cultivo
a) Nutrición microbianaMetabolismo microbiano
Macronutrientes
• Son captados por los microorganismos en cantidades relativamentegrandes.
• 95% de la célula microbiana está constituido por C, O, H, N, S, P, K, Ca,Mg y Fe.
• C, O, H, N, S y P: son componentes de carbohidratos, lípidos,proteínas y ácidos nucleicos.
a) Nutrición microbiana
Metabolismo microbianoMacronutrientes
Micronutrientes:
Factores de crecimiento
Macronutrientes
• C: Elemento más abundante en las macromoléculas
ü Constituye el 50% del peso seco de las células
ü Muchos microorganismos son heterótrofos. Necesitan algún tipo decompuesto orgánico como fuente de carbono para hacer materialcelular.
ü Los aminoácidos, ácidos grasos, ácidos orgánicos, azúcares, basesnitrogenadas, compuestos aromáticos y un sinfín de compuestosorgánicos de otro tipo pueden ser utilizados por los microorganismos.
ü Algunos microorganismos son autótrofos, capaces de construir todassus estructuras orgánicas a partir del CO2 con la energía obtenida de laluz o de compuestos inorgánicos.
a) Nutrición microbianaMetabolismo microbiano
a) Nutrición microbianaMetabolismo microbiano
N: es importante en las proteínas, en los ácidos nucleicos y en otros constituyentes celulares.
• Mayor parte del nitrógeno natural disponible está en forma inorgánica, como amoniaco (NH3), nitratos (NO3-) o N2 (puede ser usado como fuente de nitrógeno por algunas bacterias fijadoras de nitrógeno)
• La mayoría de los M.O. aprovechan NH3 y algunos nitrato.CHAPTER 6 • Molecular Biology of Bacteria 151
UN
IT 3
Cytosine (C)
NH
O
NH2
N
Thymine (T)
N
NH
O
O
H3C
Uracil(U)
N
NH
O
O
Guanine (G)
N
N
N
NH
NH2
O
N
N
65
4 321
Pyrimidine bases Purine bases
Adenine (A)
N
N
NH2
H
5
67
98
1 2
43
P
O
O–
O–O
C C
O Base
OH
H HHH C
OHRibose
Phosphate
C
CH2
H onlyin DNA
H H
H2C BaseO
O
P O–O
H H H
H
H
H2C BaseO
O
O
5′ position
P O–O
O
Nitrogen base attached to 1′ ′ position
Deoxyribose
(c)
(a)
(b)
H
H
DNA DNA only
RNA only
DNA DNA
5′
4′3′ 2′
1′
5′
4′
3′ 2′
1′
Phosphodiesterbond
3′ position
RNA RNA RNA
Figure 6.1 Components of the nucleic acids. (a) The nitrogen basesof DNA and RNA. Note the numbering system of the rings. In attachingitself to the 19 carbon of the sugar phosphate, a pyrimidine base bondsthrough N-1 and a purine base bonds at N-9. (b) Nucleotide structure.The numbers on the sugar contain a prime (9) after them because therings of the nitrogen bases are also numbered. In DNA a hydrogen ispresent on the 29-carbon of the pentose sugar. In RNA, an OH groupoccupies this position. (c) Part of a DNA chain. The nucleotides are linkedby a phosphodiester bond. In addition to the bases shown, transferRNAs (tRNAs) contain unusual pyrimidines such as pseudouracil anddihydrouracil, and various modified purines not present in other RNAs(see Figure 6.33).
Cells may be regarded as chemical machines and coding devices.As chemical machines, cells transform their vast array of macro-molecules into new cells. As coding devices, they store, process,and use genetic information. Genes and gene expression are thesubject of molecular biology. In particular, the review of molecu-lar biology in this chapter covers the chemical nature of genes,the structure and function of DNA and RNA, and the replicationof DNA. We then consider the synthesis of proteins, macromole-cules that play important roles in both the structure and thefunctioning of the cell. Our focus here is on these processes asthey occur in Bacteria. In particular, Escherichia coli, a member ofthe Bacteria, is the model organism for molecular biology and isthe main example used. Although E. coli was not the first bac-terium to have its chromosome sequenced, this organismremains the best characterized of any organism, prokaryote oreukaryote.
I DNA Structure and GeneticInformation
6.1 Macromolecules and GenesThe functional unit of genetic information is the gene. All lifeforms, including microorganisms, contain genes. Physically,genes are located on chromosomes or other large moleculesknown collectively as genetic elements. Nowadays, in the“genomics era,” biology tends to characterize cells in terms oftheir complement of genes. Thus, if we wish to understand howmicroorganisms function we must understand how genes encodeinformation.
Chemically, genetic information is carried by the nucleic acidsdeoxyribonucleic acid, DNA, and ribonucleic acid, RNA. DNAcarries the genetic blueprint for the cell and RNA is the interme-diary molecule that converts this blueprint into defined aminoacid sequences in proteins. Genetic information consists of thesequence of monomers in the nucleic acids. Thus, in contrast topolysaccharides and lipids, nucleic acids are informationalmacromolecules. Because the sequence of monomers in pro-teins is determined by the sequence of the nucleic acids thatencode them, proteins are also informational macromolecules.
The monomers of nucleic acids are called nucleotides, conse-quently, DNA and RNA are polynucleotides. A nucleotide hasthree components: a pentose sugar, either ribose (in RNA) ordeoxyribose (in DNA), a nitrogen base, and a molecule of phos-phate, PO4
3-. The general structure of nucleotides of both DNAand RNA is very similar (Figure 6.1). The nitrogen bases areeither purines (adenine and guanine) which contain two fusedheterocyclic rings or pyrimidines (thymine, cytosine, and uracil)which contain a single six-membered heterocyclic ring (Figure6.1a). Guanine, adenine, and cytosine are present in both DNAand RNA. With minor exceptions, thymine is present only inDNA and uracil is present only in RNA.
The nitrogen bases are attached to the pentose sugar by aglycosidic linkage between carbon atom 1 of the sugar and anitrogen atom in the base, either nitrogen 1 (in pyrimidine bases)or 9 (in purine bases). A nitrogen base attached to its sugar, but
NaNO3
• S: componente estructural de los aminoácidos cisteína y metionina y porque se presenta en ciertas vitaminas como la tiamina, la biotina y el ácido lipoico, así como en la coenzima A.
• La mayoría del azufre celular procede de fuentes inorgánicas, ya sean sulfatos (SO4
2-) o sulfuros (HS-).
• P: se presenta en la naturaleza en forma de fosfatos orgánicos e inorgánicos (PO43-) y la célula lo necesita fundamentalmente para la síntesis de ácidos nucleicos y fosfolípidos.
a) Nutrición microbianaMetabolismo microbiano
• K: es necesario para todos los organismos. Es cofactor de muchas enzimas.
• Ca: ayuda a estabilizar la pared bacteriana y tiene una función importante en la termorresistencia de las endosporas.
• Mg: Forma complejos con el ATP y estabiliza los ribosomas, membranas celulares y los ácidos nucleicos.
• Fe: es fundamental en la respiración celular y forma parte de los citocromos, cofactor de enzimas y de proteínas transportadoras de electrones.
• Na: es requerido por algunos microorganismos (halófilos).
a) Nutrición microbianaMetabolismo microbiano
Micronutrientes:
• Se requieren en muy pequeñas cantidades.• Son tan importantes para el funcionamiento celular como los macronutrientes.• Muchos son metales, los cuales tienen una función estructural en varias enzimas.
• Cu: es utilizado en la respiración, citocromo c oxidasa, en fotosíntesis, plastocianina y en algunas superóxido dismutasas.
• Mn: es activador de muchas enzimas, presentes en algunas superóxidosdismutasas y en la enzima que rompe la molécula de agua en fotótrofosoxigénicos.
• Zn: es requerido para la función celular de la anhidrasa carbónica, alcohol deshidrogenasa, RNA y DNA polimerasas y muchas proteínas que se unen al DNA.
a) Nutrición microbianaMetabolismo microbiano
• Factores de crecimiento:
• Son compuestos orgánicos que se necesitan en muy pequeñas cantidades y sólo por algunas células.
a) Nutrición microbianaMetabolismo microbiano
Fact
ores
de
crec
imie
nto
Vitaminas
Tiamina (B1)
Biotina
Piridoxina (B6)Aminoácidos
Purinas
Pirimidinas
Si un organismos necesita una molécula X para crecer, entonces es auxótrofo de X
Medio sintético o definido
• Son medios en que se conocen todos los componentes. Compuesto Cantidad (g·L-1)
NaNO3 1.5
K2HPO4·3H2O 0.04
MgSO4·7H2O 0.075
CaCl2·2H2O 0.036
Ácido cítrico 0.006
Citrato de amonio férrico
0.006
Na2CO3 0.02
EDTA 0.001
Solución de metales traza
1 mL·L-1
Medio BG-11 para cianobacterias
b) Medio de cultivoMetabolismo microbiano
• Los medios definidos se emplean ampliamente en investigación dado que con frecuencia se requiere conocer que esta metabolizando el m.o.
Medio complejo:
• Son medios suficientemente ricos para satisfacer las necesidades nutricionales de diversos microorganismos.
• De gran utilidad cuando se desconoce los requerimientos nutricionales de un microorganismo en particular.
Compuesto Cantidad (g·L-1)
Peptona de gelatina 1.7
Peptona de caseína 1.5
Lactosa 10
Sales biliares 1.5
Cloruro sódico 5
Rojo neutro 0.03
Cristal violeta 0.001
Agar 13.5
Medio complejo
Los medios sólidos inmovilizan a las células, permitiéndoles crecer y formar masas aisladas visibles llamadas colonias.
b) Medio de cultivoMetabolismo microbiano
Metabolismo microbiano: Introducción
¡¡¡¡Iremos desde lo general a lo específico!!!!
Existen diferentes tipos de microorganismos en función de su fuente de energía:
• Fotótrofos: utilizan la luz
• Quimiotótrofos
ü Organótrofos: compuestos orgánicos
ü Litótrofos: compuestos inorgánicos
Enzimas y energía
Biolixiviación de minerales(Solubilización de minerales por reacciones de óxido reducción)
Todo microorganismo debe ser capaz de obtener energía de compuestos de químicos o de la luz y conservar la energía como “ATP”.
¿Que es la ENERGÍA?
es la capacidad de realizar un trabajo.
ENERGÍA LIBRE es la energía liberada que es utilizable para realizar un trabajo (DGº’).
DGº’ > 0 Endotérmica, la reacción requiere energía
DGº’ < 0 Exotérmica, la reacción ocurre espontáneamente con liberación de energía; energía que la célula será capaz de conservar en forma de ATP
La energía libre nos indica sólo si en una determinada reacción se libera o se requiere energía, pero no nos indica nada acerca de la velocidad de la reacción.
Enzimas y energía
Energía libre de activación es la cantidad de energía que se requiere paraconvertir 1 mol de moléculas de sustrato (reactante) desde el estado basal (laforma estable de baja energía de la molécula) al estado de transición.
ü Una reacción no catalizada tiene una energía de activación mayor que la reacción catalizada.
ü No existe diferencia en la energía libre entre una reacción catalizada y una no catalizada (DG).
DG
Enzimas y energía
UNIT 2 • Metabolism and Growth94
Enzyme CatalysisThe catalytic power of enzymes is impressive. Enzymes increasethe rate of chemical reactions anywhere from to timesover that which would occur spontaneously. To catalyze a spe-cific reaction, an enzyme must do two things: (1) bind its sub-strate and (2) position the substrate relative to the catalyticallyactive amino acids in the enzyme’s active site. The enzyme–substrate complex (Figure 4.7) aligns reactive groups and placesstrain on specific bonds in the substrate(s). The net result is areduction in the activation energy required to make the reactionproceed from substrate(s) to product(s) (Figure 4.6). These stepsare shown in Figure 4.7 for the enzyme lysozyme, an enzymewhose substrate is the polysaccharide backbone of the bacterialcell wall polymer, peptidoglycan ( Figure 3.16).
The reaction depicted in Figure 4.6 is exergonic because thefree energy of formation of the substrates is greater than that ofthe products. Enzymes can also catalyze reactions that requireenergy, converting energy-poor substrates into energy-richproducts. In these cases, however, not only must an activationenergy barrier be overcome, but sufficient free energy must alsobe put into the reaction to raise the energy level of the substratesto that of the products. This is done by coupling the energy-requiring reaction to an energy-yielding one, such as the hydrol-ysis of ATP.
Theoretically, all enzymes are reversible in their activity. How-ever, enzymes that catalyze highly exergonic or highly endergonicreactions typically act only unidirectionally. If a particularly
1020108
exergonic or endergonic reaction needs to be reversed, a differ-ent enzyme usually catalyzes the reverse reaction.
MiniQuiz• What is the function of a catalyst? What are enzymes made of?• Where on an enzyme does the substrate bind?• What is activation energy?
III Oxidation–Reduction andEnergy-Rich Compounds
The energy released in oxidation–reduction (redox) reactionsis conserved in cells by the simultaneous synthesis of energy-
rich compounds, such as ATP. Here we first consideroxidation–reduction reactions and the major electron carrierspresent in the cell. We then examine the compounds that actuallyconserve the energy released in oxidation–reduction reactions.
4.6 Electron Donors and Electron Acceptors
An oxidation is the removal of an electron or electrons from asubstance, and a reduction is the addition of an electron or elec-trons to a substance. Oxidations and reductions are common incellular biochemistry and can involve just electrons or an elec-tron plus a proton (a hydrogen atom; H).
CH2OH CH2OH
OH
HH
H
H
HO
OH
HH
H
HH
R
O
O
O
O !(1,4)
H
OO
H
R
OH
O O
H OH
R
Active site
Substrate
O
O
O
Free lysozyme Enzyme–substratecomplex
Free lysozyme
Products
R
OH
H
CH2OH
HOH
H
CH2OH
HH H
H
Figure 4.7 The catalytic cycle of an enzyme. The enzyme depicted here, lysozyme, catalyzes thecleavage of the β-1,4-glycosidic bond in the polysaccharide backbone of peptidoglycan. Following bindingin the enzyme’s active site, strain is placed on the bond, and this favors breakage. Space-filling model oflysozyme courtesy of Richard Feldmann.
Catalizador es una sustancia que disminuye la energía de activación de una reacción y por consiguiente, aumenta la velocidad de una reacción.
ü Facilitan las reacciones pero ellos mismos no se consumen ni se modifican durante la reacción.
ü No intervienen en la energía o en el equilibrio de una reacción, sino que afectan sólo a la velocidad a las que esas reacciones ocurren.
Los catalizadores de las reacciones biológicas son proteínas llamadas enzimas.
Las enzimas son muy específicas para las reacciones que catalizan.
üEspecificidad se debe a la estructura tridimensional de la molécula de la enzima (sitio activo).
E + S è ES è E + P
Enzimas y energía
• Oxidación se define como una pérdida de un electrón o varios electrones de una sustancia.
• Reducción se define como la ganancia de un electrón (o electrones) por parte de una sustancia.
“En bioquímica Reacción redox o de oxidación-reducción transferencia no sólo de electrones sino de átomos completos de hidrógeno”.
Enzimas y energíaa) Oxidación-Reducción
Uso de energia derivada de reacciones qumicas implica rxn
Redox
H2 è 2 e- + 2 H+ Oxidación
½ O2 + 2 e- + 2 H+ èH2O Reducción_____________________H2 + ½ O2 èH2O
H2 donador de electrones y sustancia oxidada.
O2 aceptor de electrones y sustancia reducida.
•Las sustancias varían en cuanto a su tendencia a oxidarse o reducirse.
•El que se encuentre en una u otra forma dependerá de la otra pareja de sustancia oxidante y reductora.
Enzimas y energíaa) Oxidación-Reducción
Se puede establecer una escala de tendencia a la reducción, el cual se expresa como el potencial de reducción de la sustancia (E0’, en condiciones estándar pH 7).
De las dos especies, se reducirá la que tenga un mayor potencial de reducción.
§Semireacción: 2H+/H2 E0’= -0,42 V (Voltios)
§Semireacción: ½ O2/H2O E0’= +0,82 V è se reduce
Enzimas y energíaa) Oxidación-Reducción
La cantidad de energía liberada en una reacción redox depende de la naturaleza tanto del donador como del aceptor de electrones
Cuanto mayor sea la diferencia entre los respectivos potenciales de reducción, mayor será la energía liberada cuando reaccionen entre ellos.
La transferencia de electrones en una reacción oxidación-reducción en la célula, desde un donador a un aceptor, normalmente requiere uno o más intermediarios que actúan como “TRANSPORTADORES”.
Existen 2 tipos de transportadores de electrones:
1. Los que difunden libremente
Transportadores de átomos de H 2. Los que están unidos firmemente a enzimas anclados en lamembrana citoplasmática.
Enzimas y energíaa) Oxidación-Reducción
NAD+ y NADP+
citocromo c” en la cadena transportadora de e “respiración aerobia”).
coenzimas nicotinamida adeníndinucleótido (NAD+) y NAD-fosfato (NADP+).
NAD+ y NADP+ son transportadores de átomos de hidrógeno Y TRANSFIEREN 2 ÁTOMOS DE H AL SIGUIENTE TRANSPORTADOR DE UNA CADENA.
Nicotinamide
H
H
C
O
O
ONH2
H
R
+ H+
NADH + H+H
H
HC
NH2
NAD+
OCH2
OHOH
Ribose
OPHO
OCH2
OHOH
Ribose
OPHO
ON
N
N
N
NH2
Adenine
O NAD+/ NADH
E0 ′ – 0.32V
+N
N
H H2H
Figure 4.10 The oxidation–reduction coenzyme nicotinamide adeninedinucleotide (NAD1). NAD1 undergoes oxidation–reduction as shown andis freely diffusible. “R” is the adenine dinucleotide portion of NAD1.
UNIT 2 • Metabolism and Growth96
tains energy but that the chemical reaction in which the electrondonor participates releases energy. The presence of a suitableelectron acceptor is just as important as the presence of a suitableelectron donor. Lacking one or the other, the energy-releasingreaction cannot proceed. Many potential electron donors existin nature, including a wide variety of organic and inorganiccompounds.
Electron Carriers and NAD/NADH CyclingRedox reactions in microbial cells are typically mediated by oneor more small molecules. A very common carrier is the coen-zyme nicotinamide adenine dinucleotide (NAD1) (Figure 4.10).NAD1 is an electron plus proton carrier, transporting 2 e2 and 2 H1 at the same time.
The reduction potential of the NAD1/NADH couple is 20.32 V,which places it fairly high on the electron tower; that is, NADH isa good electron donor (Figure 4.10). Coenzymes such as NADHincrease the diversity of redox reactions possible in a cell by allow-ing chemically dissimilar electron donors and acceptors to inter-act, with the coenzyme acting as the intermediary. For example,electrons removed from an electron donor can reduce NAD1 toNADH, and the latter can be converted back to NAD1 by donat-ing electrons to the electron acceptor. Figure 4.11 shows an exam-ple of such electron shuttling by NAD1/NADH. In the reaction,NAD1 and NADH facilitate the redox reaction without being
Enzyme I
Enzyme II
NAD+
binding site
Active site
NADH
binding site
Activesite
Enzyme–substratecomplex
Enzyme–substratecomplex
Electron acceptorreduced(product)
NAD+
NAD+ reduction
NADH oxidation
Electron donor(substrate)
Electron donoroxidized(product)
Electron acceptor(substrate)
Enzyme I reacts with electron donor and oxidized form of coenzyme, NAD+
Enzyme II reacts with electron acceptor and reduced form of coenzyme, NADH
+
+
+
+NADH
Figure 4.11 NAD1/NADH cycling. A schematic example of redox reac-tions in two different enzymes linked by their use of either NAD1 or NADH.
As previously mentioned, all half reactions are written asreductions. However, in an actual reaction between two redoxcouples, the half reaction with the more negative E09 proceeds asan oxidation and is therefore written in the opposite direction. Inthe reaction between H2 and O2 shown in Figure 4.8, H2 is thusoxidized and is written in the reverse direction from its formalhalf reaction.
The Redox Tower and Its Relationship to DG09A convenient way of viewing electron transfer reactions in bio-logical systems is to imagine a vertical tower (Figure 4.9). Thetower represents the range of reduction potentials possible forredox couples in nature, from those with the most negative E09on the top to those with the most positive E09 at the bottom; thus,we can call the tower a redox tower. The reduced substance in theredox couple at the top of the tower has the greatest tendency todonate electrons, whereas the oxidized substance in the redoxcouple at the bottom of the tower has the greatest tendency toaccept electrons.
Using the tower analogy, imagine electrons from an electrondonor near the top of the tower falling and being “caught” byelectron acceptors at various levels. The difference in reduc-tion potential between the donor and acceptor redox couples isexpressed as DE09. The further the electrons drop from a donorbefore they are caught by an acceptor, the greater the amountof energy released. That is, is proportional to (Figure 4.9). Oxygen, at the bottom of the redox tower, is thestrongest electron acceptor of any significance in nature. In themiddle of the redox tower, redox couples can be either electrondonors or acceptors depending on which redox couples theyreact with. For instance, the 2 H1/H2 couple (20.42 V) can reactwith the fumarate/succinate (10.03 V), NO3
2/NO22 (10.42 V), or
couples, with increasing amounts of energybeing released, respectively (Figure 4.9).
Electron donors used in energy metabolism are also calledenergy sources because energy is released when they are oxidized(Figure 4.9). The point is not that the electron donor per se con-
12 O2/H2O(1 8.82 V)
DG09D E09
1. Difunden libremente
Enzimas y energíaa) Oxidación-Reducción
NAD+ y NADP+
NAD+ / NADH funciona en reacciones que generan energía (catabólicas)
NADP+ /NADPH opera fundamentalmenteen reacciones de biosíntesis (anabólicas).
A pesar que NAD+ y NADP+ tienen los mismos potenciales de reducción,generalmente funcionan en la célula en operaciones distintas.
CHAPTER 4 • Nutrition, Culture, and Metabolism of Microorganisms 97
consumed in the process. Recall that the cell requires largeamounts of a primary electron donor (the substance that was oxi-dized to yield NADH) and a final electron acceptor (such as O2).But the cell needs only a tiny amount of NAD1/NADH becausethey are constantly being recycled. All that is needed is an amountsufficient to service the redox enzymes in the cell that use thesecoenzymes in their reaction mechanisms (Figure 4.11).
NADP1/NADPH is a related redox coenzyme in which a phos-phate group is added to NAD1/NADH. NADP1/NADPH typi-cally participate in redox reactions distinct from those that useNAD1/NADH, most commonly in anabolic (biosynthetic) reac-tions in which oxidations and reductions occur.
MiniQuiz• In the reaction H2 1 O2 S H2O, what is the electron donor and
what is the electron acceptor?• Why is nitrate (NO3
2) a better electron acceptor than fumarate?• Is NADH a better electron donor than H2? Is NAD1 a better
acceptor than H1? How do you determine this?
4.7 Energy-Rich Compounds and Energy Storage
Energy released from redox reactions must be conserved by thecell if it is to be used later to drive energy-requiring cell func-tions. In living organisms, chemical energy released in redoxreactions is conserved primarily in phosphorylated compounds.The free energy released upon hydrolysis of the phosphate inthese energy-rich compounds is significantly greater than that of
12
the average covalent bond in the cell, and it is this releasedenergy that is conserved by the cell.
Phosphate can be bonded to organic compounds by either esteror anhydride bonds, as illustrated in Figure 4.12. However, not allphosphate bonds are energy-rich. As seen in the figure, the DG09of hydrolysis of the phosphate ester bond in glucose 6-phosphateis only 213.8 kJ/mol. By contrast, the DG09 of hydrolysis of thephosphate anhydride bond in phosphoenolpyruvate is 251.6kJ/mol, almost four times that of glucose 6-phosphate. Althougheither compound could be hydrolyzed to yield energy, cells typi-cally use a small group of compounds whose DG09 of hydrolysis isgreater than 230 kJ/mol as energy “currencies” in the cell. Thus,phosphoenolpyruvate is energy-rich whereas glucose 6-phosphateis not. Notice in Figure 4.12 that ATP contains three phosphates,but only two of them have free energies of hydrolysis of .30 kJ.Also notice that the thioester bond between the C and S atoms ofcoenzyme A has a free energy of hydrolysis of .30 kJ.
Adenosine TriphosphateThe most important energy-rich phosphate compound in cells isadenosine triphosphate (ATP). ATP consists of the ribonucleo-side adenosine to which three phosphate molecules are bonded inseries. ATP is the prime energy currency in all cells, being generatedduring exergonic reactions and consumed in endergonic reactions.From the structure of ATP (Figure 4.12), it can be seen that two ofthe phosphate bonds are phosphoanhydrides that have free ener-gies of hydrolysis greater than 30 kJ. Thus, the reactions ATP SADP 1 Pi and ADP S AMP 1 Pi each release roughly 32 kJ/mol ofenergy. By contrast, AMP is not energy-rich because its free energyof hydrolysis is only about half that of ADP or ATP (Figure 4.12).
UN
IT 2
N
N
N
N
OH
OCH2
NH2
OH
OP
O
OPOP–O
O–
O
O–
O
O–
P–O
O
O–
CCH2 COO–
Anhydride bond
Anhydride bonds Ester bond
PO
O
O–CH2
HCOH
HCOH
OHCH
HCOH
CHO
O–
Ester bond
Thioesterbond Anhydride bond
PO
O
O–
O–
CH3C
O
Phosphoenolpyruvate Adenosine triphosphate (ATP) (ATP) Glucose 6-phosphate
Acetyl phosphate
O
–52.0
Compound
∆G0′ > 30kJ
G0′ kJ/mol
Phosphoenolpyruvate1,3-BisphosphoglycerateAcetyl phosphateATPADP
∆G0′ < 30kJAMPGlucose 6-phosphate
–51.6
–44.8–31.8–31.8
Acetyl-CoA –35.7
–14.2–13.8
CH3 C
O
~S (CH2)2 NH
C
O
(CH2)2 NH
C
O
(CH2)3 O R
Acetyl
Acetyl-CoA
Coenzyme A
Figure 4.12 Phosphate bonds in compounds that conserve energy in bacterial metabolism. Notice,by referring to the table, the range in free energy of hydrolysis of the phosphate bonds highlighted in thecompounds. The “R” group of acetyl-CoA is a 39 phospho ADP group.
Se producen en la célula otros compuestos que pueden conservar la Eº que se libera en reacciones exotérmicas.
Por ejemplo: derivados de coenzima A (como el acetil-CoA, enlace tioéster)
Enzimas y energíab) Compuestos de alta energía
Almacenamiento de la energía, las células producen polisacáridos insolubles que luego puede oxidarse produciendo ATP.
Por ejemplo: Glucógeno, Almidón, Poli-hidroxialcanoatos “PHA”, Azufre elemental.
ü Se almacenan dentro de la célula como grandes gránulos, ü En ausencia de una fuente de energía externa, las células pueden oxidar estos
polímeros y ser capaces de hacer nuevo material celular o disponer del nivel de energía de mantenimiento.
Enzimas y energíab) Compuestos de alta energía, almacenamiento largo
plazo
Acumulación de gránulos de PHA en Rhodobacter shaeroides
Mecanismos de conservación de la energía en quimiotrofosPrincipales Rutas catabólicas
Principales Rutas catabólicas
ü Glucolisis
ü Respiración y transportadores de electrones asociados a membranas
.
QuimiotrofosMO usan compuestos químicos, como donadores de e en el metabolismo energético
1. Fermentación
2. Respiración.
mecanismosconservación de Eº:
síntesis ATP
Diferencias Fermentación Respiración
Reacciones redox ocurre en ausencia de
aceptores finales de eel O molecular o algún
otro aceptor de e, funciona como aceptor
final de e. Sintesis ATP fosforilación a nivel de
sustrato, catabolismo de un compuesto organico.
fosforilación oxidativa que produce ATP a expensa de la fuerza de protones.
UNIT 2 • Metabolism and Growth98
Although the energy released in ATP hydrolysis is 232 kJ, acaveat must be introduced here to define more precisely theenergy requirements for the synthesis of ATP. In an activelygrowing Escherichia coli cell, the ratio of ATP to ADP is about7.5:1. This deviation from equilibrium affects the energy require-ments for ATP synthesis. In such a cell, the actual energy expen-diture (that is, the ∆G, Section 4.4) for the synthesis of 1 mole ofATP is on the order of 255 to 260 kJ. Nevertheless, for the pur-poses of learning and applying the basic principles of bioenerget-ics, we assume that reactions conform to “standard conditions”(DG09), and thus we assume that the energy required for synthe-sis or hydrolysis of ATP is 32 kJ/mol.
Coenzyme ACells can use the free energy available in the hydrolysis of otherenergy-rich compounds as well as phosphorylated compounds.These include, in particular, derivatives of coenzyme A (for exam-ple, acetyl-CoA; see structure in Figure 4.12). Coenzyme A deriv-atives contain thioester bonds. Upon hydrolysis, these yieldsufficient free energy to drive the synthesis of an energy-richphosphate bond. For example, in the reactionacetyl-S-CoA 1 H2O 1 ADP 1 Pi S
acetate2 1 HS-CoA 1 ATP 1 H1
the energy released in the hydrolysis of coenzyme A is conservedin the synthesis of ATP. Coenzyme A derivatives (acetyl-CoA isjust one of many) are especially important to the energetics ofanaerobic microorganisms, in particular those whose energymetabolism depends on fermentation. We return to the impor-tance of coenzyme A derivatives many times in Chapter 14.
Energy StorageATP is a dynamic molecule in the cell; it is continuously beingbroken down to drive anabolic reactions and resynthesized at theexpense of catabolic reactions. For longer-term energy storage,microorganisms produce insoluble polymers that can be catabo-lized later for the production of ATP.
Examples of energy storage polymers in prokaryotes includeglycogen, poly-β-hydroxybutyrate and other polyhydroxyalka-noates, and elemental sulfur, stored from the oxidation of H2S bysulfur chemolithotrophs. These polymers are deposited withinthe cell as large granules that can be seen with the light or elec-tron microscope ( Section 3.10). In eukaryotic microorga-nisms, polyglucose in the form of starch and lipids in the form ofsimple fats are the major reserve materials. In the absence of anexternal energy source, a cell can break down these polymers tomake new cell material or to supply the very low amount ofenergy, called maintenance energy, needed to maintain cellintegrity when it is in a nongrowing state.
MiniQuiz• How much energy is released per mole of ATP converted to
ADP 1 Pi under standard conditions? Per mole of AMP con-verted to adenosine and Pi?
• During periods of nutrient abundance, how can cells prepare forperiods of nutrient starvation?
IV Essentials of Catabolism
Two series of reactions—fermentation and respiration—arelinked to energy conservation in chemoorganotrophs:
Fermentation is the form of anaerobic catabolism in which anorganic compound is both an electron donor and an electronacceptor, and ATP is produced by substrate-level phosphoryla-tion; and respiration is the catabolism in which a compound isoxidized with O2 (or an O2 substitute) as the terminal electronacceptor, usually accompanied by ATP production by oxidativephosphorylation. In both series of reactions, ATP synthesis iscoupled to energy released in oxidation–reduction reactions.
One can look at fermentation and respiration as alternativemetabolic choices available to some microorganisms. In orga-nisms that can both ferment and respire, such as yeast, fermenta-tion is necessary when conditions are anoxic and terminalelectron acceptors are absent. When O2 is available, respirationcan take place. We will see that much more ATP is produced inrespiration than in fermentation and thus respiration is the pre-ferred choice (see the Microbial Sidebar, “Yeast Fermentation, thePasteur Effect, and the Home Brewer”). But many microbial habi-tats lack O2 or other electron acceptors that can substitute for O2in respiration (see Figure 4.22), and in such habitats, fermentationis the only option for energy conservation by chemoorganotrophs.
4.8 GlycolysisIn fermentation, ATP is produced by a mechanism calledsubstrate-level phosphorylation. In this process, ATP is syn-thesized directly from energy-rich intermediates during steps inthe catabolism of the fermentable substrate (Figure 4.13a). This
Substrate-level phosphorylation
Oxidative phosphorylation
Less energizedmembrane
Energizedmembrane
IntermediatesEnergy-richintermediates
ATP
(a)
(b)
C~PB~PBA
Pi
D
ADP ATP
ADP + Pi
++
++ +
+
+++ + + + + + + + + + +++ + + ++
+
+++
+++
++
+
+++ + + + + + + + +++ + + +
––––
–––– – – – – – – – – ––– – – ––
––––
––
––– – – – – – – ––– – – –
++
+
++ + + + + + + ++
++
+
+
+
++ + + + ++ +
––
–– – – – – – ––––
–
–– – – ––– –
Figure 4.13 Energy conservation in fermentation and respiration.(a) In fermentation, substrate-level phosphorylation produces ATP. (b) In respi-ration, the cytoplasmic membrane, energized by the proton motive force, dis-sipates energy to synthesize ATP from ADP 1 Pi by oxidative phosphorylation.
Mecanismos de conservación de la energía
(a)En la fermentación, la síntesis de ATP tiene lugar como resultado de una fosforilación a nivel de sustrato.
(a)En la respiración, la membrana citoplasmática está energéticamente activada por la fuerza motriz de protones y pierde parte de esa energía en la formación de ATP a partir de ADP y fosfato inorgánico (Pi), en el proceso llamado fosforilación oxidativa.
Rutas catabólicas
Glucolisis, ruta bioquímica utilizada para la fermentación de la glucosa, también denominada vía de Embden-Meyerhof.
UNIT 2 • Metabolism and Growth100
H
Stage I
Stage II
Stage III
Intermediates
Energetics
2 Pyruvate
2 lactate
2 ethanol + 2 CO2
+ 2 NADH2 ATP
ATP ATP
2 ATPPyruvate
OCH2
C O
H2COH 2 NAD+
HC O
HC
H2CO
C
CO
O
CH3
C
CO
O
CH2
C
C
O
O
CH2HO
C
COH
O
O
CH2
O
H
C
COH
O
OCH2
HOCH2
O
OHHOH
HH
HO
H OH
H
OCH2
O
OHHOH
OH
HH
H OH
H
H
OOCH2
OHOH
H2COH
H HOH H
OOCH2
HHO
H2CO
H HOOH
Glucose
A
I
B
H
C
G
D
E
F
Glucose 6-P
Glucose 2 ethanol +
Glucose 2 lactate
2 CO2
A
Fructose 6-PB
Fructose 1, 6-PC
Dihydroxyacetone-PD
Glyceraldehyde-3-PE
Enzymes
Hexokinase1
Isomerase2
Phosphofructokinase3
Aldolase4
Triosephosphate isomerase5
5
6
78910
1 2 3 4
11
12 13
7 Phosphoglycerokinase
8 Phosphoglyceromutase
9 Enolase
10 Pyruvate kinase
11
Glyceraldehyde-3-Pdehydrogenase
6
Pyruvate decarboxylase
Lactate dehydrogenase
12
Alcohol dehydrogenase13
1, 3-BisphosphoglycerateF
3-P-GlycerateG
2-P-GlycerateH
PhosphoenolpyruvateI
–196 kJ
Yeast
Lactic acid bacteria
–239 kJ
P P
PPP
P P
P P
P
P
2 2 2 2 2O–O– O– O–
OH
Figure 4.14 Embden–Meyerhof–Parnas pathway (glycolysis). The sequence of reactions in the catab-olism of glucose to pyruvate and then on to fermentation products. Pyruvate is the end product of glycolysis,and fermentation products are made from it. The blue table at the bottom left lists the energy yields from thefermentation of glucose by yeast or lactic acid bacteria.
is in contrast to oxidative phosphorylation, typical of respira-tion, in which ATP is produced at the expense of the protonmotive force (Figure 4.13b).
The fermentable substrate in a fermentation is both the electrondonor and electron acceptor; not all compounds can be fermented,but sugars, especially hexoses such as glucose, are excellent fer-mentable substrates. A common pathway for the catabolism ofglucose is glycolysis, which breaks down glucose into pyruvate.Glycolysis is also called the Embden–Meyerhof–Parnas pathwayfor its major discoverers. Whether glucose is fermented orrespired, it travels through this pathway. Here we focus on thereactions of glycolysis and the reactions that follow under anoxicconditions.
Glycolysis can be divided into three stages, each involving aseries of enzymatic reactions. Stage I comprises “preparatory”reactions; these are not redox reactions and do not release energybut instead lead to the production of a key intermediate of the
pathway. In Stage II, redox reactions occur, energy is conservedin the form of ATP, and two molecules of pyruvate are formed.The reactions of glycolysis are finished at this point. However,redox balance has not yet been achieved. So, in Stage III, redoxreactions occur once again and fermentation products areformed (Figure 4.14).
Stage I: Preparatory ReactionsIn Stage I glucose is phosphorylated by ATP, yielding glucose 6-phosphate; the latter is then isomerized to fructose 6-phosphate.A second phosphorylation leads to the production of fructose1,6-bisphosphate. The enzyme aldolase then splits fructose 1,6-bisphosphate into two 3-carbon molecules, glyceraldehyde 3-phosphate and its isomer, dihydroxyacetone phosphate, whichcan be converted into glyceraldehyde 3-phosphate. To this point,all of the reactions, including the consumption of ATP, have pro-ceeded without redox reactions.
ETAPA I: preparacion-Serie de reacciones que no implican ni oxidación ni reducción y que no liberan energía.-Conducen a la producción a partir de glucosa de dos moléculas de gliceraldehído-3-PETAPA II: -Ocurre un proceso la energía se conserva en forma de ATP y se forman “moléculas de piruvato.
ETAPA III: -Una segunda reacción redoxy se originan los productos de fermentación (etanol y CO2 o ácido láctico).
Rutas catabólicas
UNIT 2 • Metabolism and Growth100
H
Stage I
Stage II
Stage III
Intermediates
Energetics
2 Pyruvate
2 lactate
2 ethanol + 2 CO2
+ 2 NADH2 ATP
ATP ATP
2 ATPPyruvate
OCH2
C O
H2COH 2 NAD+
HC O
HC
H2CO
C
CO
O
CH3
C
CO
O
CH2
C
C
O
O
CH2HO
C
COH
O
O
CH2
O
H
C
COH
O
OCH2
HOCH2
O
OHHOH
HH
HO
H OH
H
OCH2
O
OHHOH
OH
HH
H OH
H
H
OOCH2
OHOH
H2COH
H HOH H
OOCH2
HHO
H2CO
H HOOH
Glucose
A
I
B
H
C
G
D
E
F
Glucose 6-P
Glucose 2 ethanol +
Glucose 2 lactate
2 CO2
A
Fructose 6-PB
Fructose 1, 6-PC
Dihydroxyacetone-PD
Glyceraldehyde-3-PE
Enzymes
Hexokinase1
Isomerase2
Phosphofructokinase3
Aldolase4
Triosephosphate isomerase5
5
6
78910
1 2 3 4
11
12 13
7 Phosphoglycerokinase
8 Phosphoglyceromutase
9 Enolase
10 Pyruvate kinase
11
Glyceraldehyde-3-Pdehydrogenase
6
Pyruvate decarboxylase
Lactate dehydrogenase
12
Alcohol dehydrogenase13
1, 3-BisphosphoglycerateF
3-P-GlycerateG
2-P-GlycerateH
PhosphoenolpyruvateI
–196 kJ
Yeast
Lactic acid bacteria
–239 kJ
P P
PPP
P P
P P
P
P
2 2 2 2 2O–O– O– O–
OH
Figure 4.14 Embden–Meyerhof–Parnas pathway (glycolysis). The sequence of reactions in the catab-olism of glucose to pyruvate and then on to fermentation products. Pyruvate is the end product of glycolysis,and fermentation products are made from it. The blue table at the bottom left lists the energy yields from thefermentation of glucose by yeast or lactic acid bacteria.
is in contrast to oxidative phosphorylation, typical of respira-tion, in which ATP is produced at the expense of the protonmotive force (Figure 4.13b).
The fermentable substrate in a fermentation is both the electrondonor and electron acceptor; not all compounds can be fermented,but sugars, especially hexoses such as glucose, are excellent fer-mentable substrates. A common pathway for the catabolism ofglucose is glycolysis, which breaks down glucose into pyruvate.Glycolysis is also called the Embden–Meyerhof–Parnas pathwayfor its major discoverers. Whether glucose is fermented orrespired, it travels through this pathway. Here we focus on thereactions of glycolysis and the reactions that follow under anoxicconditions.
Glycolysis can be divided into three stages, each involving aseries of enzymatic reactions. Stage I comprises “preparatory”reactions; these are not redox reactions and do not release energybut instead lead to the production of a key intermediate of the
pathway. In Stage II, redox reactions occur, energy is conservedin the form of ATP, and two molecules of pyruvate are formed.The reactions of glycolysis are finished at this point. However,redox balance has not yet been achieved. So, in Stage III, redoxreactions occur once again and fermentation products areformed (Figure 4.14).
Stage I: Preparatory ReactionsIn Stage I glucose is phosphorylated by ATP, yielding glucose 6-phosphate; the latter is then isomerized to fructose 6-phosphate.A second phosphorylation leads to the production of fructose1,6-bisphosphate. The enzyme aldolase then splits fructose 1,6-bisphosphate into two 3-carbon molecules, glyceraldehyde 3-phosphate and its isomer, dihydroxyacetone phosphate, whichcan be converted into glyceraldehyde 3-phosphate. To this point,all of the reactions, including the consumption of ATP, have pro-ceeded without redox reactions.
Glucosa Glucosa-6P Fructosa-6P Fructosa-1,6P Dihidroxiacetona-P
Gliceraldehído-3P
Ácido 1,3-difosfoglicérico
Piruvato3P-Glicerato2P-Glicerato
Ácido 1,3-fosfoglicérico
Glucolisis
Rutas catabólicas
FermentaciónRutas catabólicas
Respiración y transportadores de electrones asociados a membranas
(1) el modo en que los e son transferidosdesde el compuesto orgánico hasta elaceptor final de electrones, originandosíntesis de ATP a expensas de la fuerzamotriz de protones a través de lamembrana.
(2) las vías bioquímicas implicadas en latransformación del carbono orgánico a CO2.
El proceso por el que un compuesto se oxida usando O2 como aceptor final de
electrones se llama RESPIRACIÓN AERÓBICA. La respiración aeróbica se centra en:
Conservación de la energía a partir de la
fuerza motriz de protones
Flujo de carbono en la respiración: ciclo del ácido
cítrico
Rutas catabólicas
Conservación de la energía a partir de la fuerza motriz de protones respiración aerobica
CHAPTER 4 • Nutrition, Culture, and Metabolism of Microorganisms 103
flavoproteins, quinones accept 2 e2 1 2 H1 but transfer only 2 e2 to the next carrier in the chain; quinones typically participateas links between iron–sulfur proteins and the first cytochromesin the electron transport chain.
MiniQuiz• In what major way do quinones differ from other electron carriers
in the membrane?• Which electron carriers described in this section accept
2 e2 1 2 H1? Which accept electrons only?
4.10 The Proton Motive ForceThe conservation of energy by oxidative phosphorylation islinked to an energized state of the membrane (Figure 4.13b). Thisenergized state is established by electron transport reactionsbetween the electron carriers just discussed. To understand howelectron transport is linked to ATP synthesis, we must firstunderstand how the electron transport system is oriented in thecytoplasmic membrane. Electron transport carriers are orientedin the membrane in such a way that, as electrons are transported,protons are separated from electrons. Two electrons plus twoprotons enter the electron transport chain from NADH throughNADH dehydrogenase to initiate the process. Carriers in theelectron transport chain are arranged in the membrane in orderof their increasingly positive reduction potential, with the finalcarrier in the chain donating the electrons plus protons to a ter-minal electron acceptor such as O2 (Figure 4.19).
During electron transport, H1 are extruded to the outer sur-face of the membrane. These H1 originate from two sources: (1)NADH and (2) the dissociation of water (H2O) into H1 and OH2
in the cytoplasm. The extrusion of H1 to the environment resultsin the accumulation of OH2 on the inside of the membrane.However, despite their small size, neither H1 nor OH2 can dif-fuse through the membrane because they are charged (Section 3.4). As a result of the separation of H1 and OH2, thetwo sides of the membrane differ in both charge and pH.
UN
IT 2
C
C
CCH3O C
C
O
(CH2
CH3
CH C
CH3
CH2)nH
Oxidized
Reduced
O
CH3O C
C
C
C
C
OH
CH3
OH
R
2H
CH3O C
CH3O C
Figure 4.18 Structure of oxidized and reduced forms of coenzymeQ, a quinone. The five-carbon unit in the side chain (an isoprenoid)occurs in a number of multiples, typically 6–10. Oxidized quinone requires2 e2 and 2 H1 (2 H) to become fully reduced (dashed red circles).
4 H+
NAD+
QH2
2 H+
4 H+2 H+
4 H+ + O2
Complex I
NADH + H+
FADH2
Fumarate
Succinate
FAD
Complex III
Complex IV
CYTOPLASMEN
VIR
ON
ME
NT
+0.39
+0.36
+0.1
0.0
–0.22
E0′(V)
E0′(V)
c1
Fe/S
FMNFe/S
2 e–
e–
e–e–
e–
e–
+
++
+
+
+
+
+
+
+
+
+
+
+
+
+
+
––
–
––
––
––
–
–
––
–
cyt acyt a3
e–
cytbL
bHe–
cytc H2O
Q
Qcycle
Complex II
–0.32 V
–0.22 V
+0.82 V
2 H+
12
Figure 4.19 Generation of the proton motive force during aerobicrespiration. The orientation of electron carriers in the membrane ofParacoccus denitrificans, a model organism for studies of respiration. The1 and – charges at the edges of the membrane represent H1 and OH2,respectively. values for the major carriers are shown. Note how whena hydrogen atom carrier (for example, FMN in Complex I) reduces anelectron-accepting carrier (for example, the Fe/S protein in Complex I),protons are extruded to the outer surface of the membrane. Abbrevia-tions: FMN, flavin mononucleotide; FAD, flavin adenine dinucleotide; Q,quinone; Fe/S, iron–sulfur–protein; cyt a, b, c, cytochromes (bL and bH,low- and high-potential b-type cytochromes, respectively). At the quinonesite, electrons are recycled during the “Q cycle.” This is because elec-trons from QH2 can be split in the bc1 complex (Complex III) between theFe/S protein and the b-type cytochromes. Electrons that travel throughthe cytochromes reduce Q (in two, one-electron steps) back to QH2, thusincreasing the number of protons pumped at the Q-bc1 site. Electronsthat travel to Fe/S proceed to reduce cytochrome c1, then cytochrome c,and then a-type cytochromes in Complex IV, eventually reducing O2 toH2O (2 electrons and 4 protons are required to reduce O2 to H2O alongwith 2 H1 extruded, and these come from electrons through cyt c andcytoplasmic protons, respectively). Complex II, the succinate dehydroge-nase complex, bypasses Complex I and feeds electrons directly into thequinone pool at a more positive than NADH (see the electron tower inFigure 4.9).
E09
12
E09
Rutas catabólicas
Glucosa
ATP
ADP
G6P F-1,6P2x NAD+
2x NADH
2x 1,3DFG2x ADP
2x ATP
2x 3-Pglicerato
2x 2-Pglicerato2x ADP
2x ATP
2x Piruvato2x NADH2x NAD+
2x Etanol
2x CO2
(2 pasos)
(2 pasos)
(3 pa sos)
(2 pasos)
(2 pasos)
ATP
ADP
2x isocitrato
2x α-cetoglutarato
2x succinil-coA2x succinato
2x fumarato
2x oxalacetato 2x NADP+
2x NADPH
2x CoA + 2x NAD+
2x NADH
2x GDP2x GTP
2x FADH2
2x FAD
2x NADH
2x NAD+2x CO2
2x CO2
2x CoA+2x NAD+
2x NADH
2x CoA
2x Acetil-CoA2x CoA
½ O2+1 NADH + 11 H+int →1 NAD++10 H+
ext+H2O
½ O2+1 FADH2 + 6 H+int →1 FAD+6H+
ext+H2O
1 ADP+ Pi+4H+ext →1 ATP+4 H+
int
Reacciones en cadena transportadora de electrones
ATP-asa
1 NADPH ↔ 1 NADH
Interconversiones energéticas
1 GTP ↔ 1 ATP
Fosforilación oxidativa en presencia de oxígeno
2x CO2
2 Carbonos
3 Carbonos
4 Carbonos
5 Carbonos
6 Carbonos
G6P: glucosa 6 fosfatoF-1,6P: fructosa 6 fosfato1,3 DFG: 1,3 difosfoglicerato3-Pglicerato: 3 fosfoglicerato2-Pglicerato: 2 fosfoglicerato
Ciclo de Krebs
+ y – = H+ y OH-, FMN= flavoproteína; Q, quinona; Fe/S= proteína con hierro y azufre;cit a,b,c= citocromos
I: NADH deshidrogenasa
II: Succinato-Coenzima Q reductasa
III: Complejo bc1
IV: citocromo c oxidasa
Enzimas y energíac) Rutas catabólicas
Conservación de la energía a partir de la fuerza motriz de protones
CHAPTER 4 • Nutrition, Culture, and Metabolism of Microorganisms 105
Reversibility of ATPaseATPase is reversible. The hydrolysis of ATP supplies torque for γεto rotate in the opposite direction from that in ATP synthesis,and this catalyzes the pumping of H1 from the inside to the out-side of the cell through Fo. The net result is generation instead ofdissipation of the proton motive force. Reversibility of theATPase explains why strictly fermentative organisms that lackelectron transport chains and are unable to carry out oxidativephosphorylation still contain ATPases. As we have said, manyimportant reactions in the cell, such as motility and transport,require energy from the pmf rather than from ATP. Thus,ATPase in organisms incapable of respiration, such as the strictlyfermentative lactic acid bacteria, for example, functions unidirec-tionally to generate the pmf necessary to drive these importantcell functions.
MiniQuiz• How do electron transport reactions generate the proton motive
force?• What is the ratio of H1 extruded per NADH oxidized through the
electron transport chain of Paracoccus shown in Figure 4.19? At which sites in the chain is the proton motive force beingestablished?
• What structure in the cell converts the proton motive force toATP? How does it function?
4.11 The Citric Acid CycleNow that we have a grasp of how ATP is made in respiration, weneed to consider the important reactions in carbon metabolismassociated with formation of ATP. Our focus here is on the citricacid cycle, also called the Krebs cycle, a key pathway in virtually all cells.
Respiration of GlucoseThe early biochemical steps in the respiration of glucose are thesame as those of glycolysis; all steps from glucose to pyruvate(Figure 4.14) are the same. However, whereas in fermentationpyruvate is reduced and converted into products that areexcreted, in respiration pyruvate is oxidized to CO2. The pathwayby which pyruvate is completely oxidized to CO2 is called thecitric acid cycle (CAC), summarized in Figure 4.21.
Pyruvate is first decarboxylated, leading to the production ofCO2, NADH, and the energy-rich substance acetyl-CoA (Figure4.12). The acetyl group of acetyl-CoA then combines with thefour-carbon compound oxalacetate, forming the six-carbon com-pound citric acid. A series of reactions follow, and two additionalCO2 molecules, three more NADH, and one FADH are formed.Ultimately, oxalacetate is regenerated to return as an acetylacceptor, thus completing the cycle (Figure 4.21).
CO2 Release and Fuel for Electron TransportThe oxidation of pyruvate to CO2 requires the concerted activityof the citric acid cycle and the electron transport chain. For eachpyruvate molecule oxidized through the citric acid cycle, threeCO2 molecules are released (Figure 4.21). Electrons released dur-ing the oxidation of intermediates in the citric acid cycle aretransferred to NAD1 to form NADH, or to FAD to form FADH2.This is where respiration and fermentation differ in a major way.Instead of being used in the reduction of pyruvate as in fermenta-tion (Figure 4.14), in respiration, electrons from NADH andFADH2 are fuel for the electron transport chain, ultimately result-ing in the reduction of an electron acceptor (O2) to H2O. Thisallows for the complete oxidation of glucose to CO2 along with amuch greater yield of energy. Whereas only 2 ATP are producedper glucose fermented in alcoholic or lactic acid fermentations(Figure 4.14), a total of 38 ATP can be made by aerobically respir-ing the same glucose molecule to CO2 1 H2O (Figure 4.21b).
UN
IT 2
F1
FoFo
c12
cc aaa
Out
b2 b2
In
F1
Out
In
H+H+H+
α
α
α
α
β β
β
γ γ
δδ
ε
ε
Membrane
(a) (b)
Sie
gfrie
d En
gelb
rech
t-Va
ndré
ATP
ADP + Pi
Figure 4.20 Structure and function of ATPsynthase (ATPase) in Escherichia coli. (a)Schematic. F1 consists of five different polypeptidesforming an α3β3γεδ complex, the stator. F1 is thecatalytic complex responsible for the interconver-sion of ADP 1 Pi and ATP. Fo, the rotor, is integratedin the membrane and consists of three polypep-tides in an ab2c12 complex. As protons enter, thedissipation of the proton motive force drives ATPsynthesis (3 H1/ATP). ATPase is reversible in thatATP hydrolysis can drive formation of a protonmotive force. (b) Space-filling model. The color-coding corresponds to the art in part a. Since pro-ton translocation from outside the cell to inside thecell leads to ATP synthesis by ATPase, it followsthat proton translocation from inside to outside inthe electron transport chain (Figure 4.19) repre-sents work done on the system and a source ofpotential energy.
Fosforilación oxidativa (sistemas respiratorios) o fotofosforilación (organismos fototróficos).
La fuerza motriz de protones es convertida en ATP a través de un complejo enzimático situado en la membrana llamado ATP sintetasa o ATPasa
Depende de ATP sintetasa o ATPasa:
•F1 pieza globular, formada por varias subunidades.•F0 canal conductor de protones•Atraviesa la membrana mitocondrial
•Por 1 ATP producido se requiere 3-4 H+
•Acción de ATPasa es reversible
Enzimas y energíac) Rutas catabólicas
Conservación de la energía a partir de la fuerza motriz de protones
Flujo de carbono en la Respiración: Ciclo del ácido cítrico “CAC”UNIT 2 • Metabolism and Growth106
(1) Glycolysis: Glucose + 2 NAD+
(a) Substrate-level phosphorylation 2 ADP + Pi 2 ATP(b) Oxidative phosphorylation 2 NADH 6 ATP
2 Pyruvate– + 2 ATP + 2 NADH
to CAC
8 ATP
to Complex I
Energetics Balance Sheet for Aerobic Respiration
(a) (b)
3 CO2 + 4 NADH + FADH2 + GTP(2) CAC: Pyruvate– + 4 NAD+ + GDP + FAD
(3) Sum: Glycolysis plus CAC 38 ATP per glucose
(a) Substrate-level phosphorylation 1 GDP + Pi 1 GTP (=1 ATP) (b) Oxidative phosphorylation 4 NADH 12 ATP 1 FADH2 2 ATP
15 ATP ( 2)
to Complex IIto Complex I
NAD(P)H
NAD+ + CoA
CoA
Oxalacetate2– Citrate3–
Aconitate3–
Isocitrate3–
Malate2–
NADH
NAD+
Fumarate2–
Succinate2–
Succinyl-CoA
FADH2
FAD
CoAGTP GDP + Pi NADH
CoA + NAD+
NAD(P)+
Acetyl-CoA
Pyruvate– (three carbons)
C2C4C5C6
_-Ketoglutarate2–
NADH
CO2
CO2
CO2
Figure 4.21 The citric acid cycle. (a) The citric acid cycle (CAC) begins when the two-carbon compoundacetyl-CoA condenses with the four-carbon compound oxalacetate to form the six-carbon compound citrate.Through a series of oxidations and transformations, this six-carbon compound is ultimately converted back tothe four-carbon compound oxalacetate, which then begins another cycle with addition of the next moleculeof acetyl-CoA. (b) The overall balance sheet of fuel (NADH/FADH2) for the electron transport chain and CO2generated in the citric acid cycle. NADH and FADH2 feed into electron transport chain Complexes I and II,respectively (Figure 4.19).
Biosynthesis and the Citric Acid CycleBesides playing a key role in catabolism, the citric acid cycle playsanother important role in the cell. The cycle generates several keycompounds, small amounts of which can be drawn off for biosyn-thetic purposes when needed. Particularly important in thisregard are α-ketoglutarate and oxalacetate, which are precursorsof several amino acids (Section 4.14), and succinyl-CoA, needed toform cytochromes, chlorophyll, and several other tetrapyrrole com-pounds (Figure 4.16). Oxalacetate is also important because it canbe converted to phosphoenolpyruvate, a precursor of glucose. Inaddition, acetate provides the starting material for fatty acid biosyn-thesis (Section 4.15, and see Figure 4.27). The citric acid cycle thusplays two major roles in the cell: bioenergetic and biosynthetic.Much the same can be said about the glycolytic pathway, as certainintermediates from this pathway are drawn off for various biosyn-thetic needs as well (Section 4.13).
MiniQuiz• How many molecules of CO2 and pairs of electrons are released
per pyruvate oxidized in the citric acid cycle?• What two major roles do the citric acid cycle and glycolysis have
in common?
4.12 Catabolic DiversityThus far in this chapter we have dealt only with catabolism bychemoorganotrophs. We now briefly consider catabolic diver-sity, some of the alternatives to the use of organic compounds aselectron donors, with emphases on both electron and carbonflow. Figure 4.22 summarizes the mechanisms by which cellsgenerate energy other than by fermentation and aerobic respira-tion. These include anaerobic respiration, chemolithotrophy, andphototrophy.
Anaerobic RespirationUnder anoxic conditions, electron acceptors other than oxygencan be used to support respiration in certain prokaryotes. Theseprocesses are called anaerobic respiration. Some of the electronacceptors used in anaerobic respiration include nitrate (NO3
2,reduced to nitrite, NO2
2, by Escherichia coli or to N2 byPseudomonas species), ferric iron (Fe31, reduced to Fe21 byGeobacter species), sulfate (SO4
22, reduced to hydrogen sulfide,H2S, by Desulfovibrio species), carbonate (CO3
22, reduced tomethane, CH4, by methanogens or to acetate by acetogens), andeven certain organic compounds. Some of these acceptors, forexample Fe31, are often only available in the form of insoluble
ü Una ruta importante para la oxidación total del piruvato a CO2 es el llamado ciclo del ácido cítrico (CAC).
ü Por cada molécula de piruvato que se oxida en el ciclo se producen tres de CO2. 1:3
ü Electrones liberados durante la oxidación de intermediarios del CAC son transferidos a enzimas que contienen NAD+ o FAD.
ü a-cetoglutarato y oxalacetato son precursores de ciertos aminoácidos y el succinil-CoA que se necesita para formar el anillo porfirínico de los citocromos.
ü Oxalacetato puede convertirse en fosfoenolpiruvato, un precursor a su vez de la glucosa.
ü Acetil-CoA representa el material de origen para la biosíntesis de ácidos grasos.
Rutas catabólicas Fermentación; piruvato à productos de fermentación.
Respiración; piruvato à se oxida por completo a CO2.
Respiración y transportadores de electrones asociados a membranas
Rutas catabólicas
ATP ?
• La energía para el anabolismo la suministra el ATP o la fuerza motriz de protones.
• Energía generada durante el catabolismo se consume tanto en la formación de monómeros como durante la polimerización de los monómeros para formar las respectivas macromoléculas.
d)Rutas anabólicas
Podemos distinguir dos tipos de anabolismo
•Anabolismo autótrofo: consiste en la síntesis de moléculas orgánicas sencillas a partir de precursores inorgánicos tales como el CO2, el H2O y el NH3.Solamente pueden realizarlo las células autótrofas.
Ejemplos:ü Fotosíntesis, que utiliza la energía de la luz (en las células fotolitótrofas)ü Quimiosíntesis, que utiliza la energía liberada en reacciones redox (en las células quimiolitótrofas).
•Anabolismo heterótrofo: consiste en la síntesis de moléculas orgánicas progresivamente más complejas a partir de moléculas orgánicas más sencillas. Todas las células pueden llevarlo a cabo (también las autótrofas).
Rutas anabólicas
Bibliografía
§ Brock Biología de los microorganismosCapítulos 5Michael T. MadiganJohn M . MartinkoPaul V. DunlapDavid P. Clark
§ Animacioneshttp://highered.mheducation.com/sites/0072556781/student_view0/chapter9/animation_quiz.html
