Molecular biophysics lecture notes

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INTRODUCTION TO MOLECULAR BIOPHYSICS © 2003 by CRC Press LLC1 Origins and Evolution of Life 1.1 Initiation Planet Earth was formed about 4.6 billion years ago as a result of accretions (inelastic collisions and agglomerations) of larger and larger rocky fragments formed gradually from the dust component of the gaseous dusty cloud that was the original matter of the Solar System. The Great Bombardment ended only 3.9 billion years ago when a stream of meteorites falling onto the surface of the newly formed planet reached a more or less constant intensity. The first well preserved petrified microstamps of relatively highly organized living organisms similar to today’s cyanobacteria emerged about 3.5 billion years ago (Schopf, 1999), so life on Earth must have developed within the relatively short span of a few hundred million years. Rejecting the hypothesis of an extraterrestrial origin of life, not so much for rational as for emotional reasons, we have to answer the question of the origins of the simplest elements of living organisms: amino acids, simple sugars (monosaccharides) and nitrogenous bases. Three equally probable hypotheses have been put forward to explain their appearance (Orgel, 1998). According to the first and the oldest theory, these compounds resulted from electric discharges and ultraviolet irradiation of the primary Earth atmosphere containing mostly CO (as the atmospheres of Mars and 2 Venus do today), H O, and strongly reducing gases (CH ,NH , and H S). According 2 4 3 2 to the second hypothesis, the basic components of living organisms were formed in space outside the orbits of large planets and transferred to the Earth’s surface via collisions with comets and indirectly via carbon chondrites. The third hypothesis is that these compounds appeared at the oceanic rifts where the new Earth’s crust was ◦ formed and where water overheated to 400 C containing strongly reducing FeS, H , 2 and H S met cool water containing CO . 2 2 The origin issue is still open and all three hypotheses have been seriously criticized. First, the primary Earth atmosphere might not have been reducing strongly enough. Second, organic compounds from outer space may have deteriorated while passing through the Earth’s atmosphere. Third, the reduction of CO in oceanic rifts requires 2 nontrivial catalysts. The three most important characteristics of life that distinguish it from other natural phenomena were expressed by Charles Darwin, whose theory of evolution is so crucial to modern biology (Dawkins, 1986). Taking into account the achievements of post Darwinian genetics and biochemistry, we define life as a process characterized by con- tinuous (1) reproduction, (2) variability, and (3) selection (survival of the fittest). An 1 © 2003 by CRC Press LLC2 Introduction to Molecular Biophysics (a) replication transcription translation DNA RNA PROTEIN replication (b) in RNA-viruses prions PROTEIN DNA RNA reverse transcription in retroviruses FIGURE 1.1 Processing genetic information. (a) The classical dogma. The information is carried by DNA which undergoes replication during biological reproduction and transcription into RNA when it is to be expressed; gene expression consists oftranslationoftheinformationwritteninRNAontoaparticularproteinstruc- ture. (b) Modern version of the classical dogma. RNA can be replicated and transcribedintheoppositedirectionintoDNA.Proteinsalsocancarryinforma- tion as is assumed to occur in prion diseases. individual must have a replicable and modifiable program, proper metabolism (a mechanism of matter and energy conversion), and capability of self-organization to maintain life. The emergence of molecular biology in the 1950s answered many questions about the structures and functioning of the three most important classes of biological macro- molecules: DNA (deoxyribonucleic acid), RNA (ribonucleic acid), and proteins. However, in the attempts to develop a possible scenario of evolution from small organic particles to large biomolecules, a classical chicken-and-egg question was encountered: what appeared first? The DNA that carried the coded information on enzymatic proteins controlling the physiological processes that determined the fitness of an individual or the proteins that enabled the replication of DNA, its transcription into RNA, and the translation of certain sequences of amino acids into new proteins? See Figure 1.1a for illustration. This question was resolved in the 1970s as a result of the evolutionary experimen- tation in Manfred Eigen’s laboratory (Biebricher and Gardiner, 1997). The primary macromolecular system undergoing Darwin’s evolution may have been RNA. Single- stranded RNA is not only the information carrier, program, or genotype. Because of a specific spatial structure, RNA is also an object of selection or a phenotype. Equipped with the concept of a hypercycle (Eigen and Schuster, 1977) and inspired by Sol Spigelman, Eigen used virial RNA replicase (Figure 1.1b), a protein, to pro- duce new generations of RNA in vitro. The complementary RNA could polymerize spontaneously, without replicase, using the matrix of the already existing RNA as a template. Consequently, we can imagine a very early “RNA world” composed only of © 2003 by CRC Press LLCOrigins and Evolution of Life 3 nucleotides, their phosphates, and their polymers — subject to Darwinian evolution, and thus alive based on the definition adopted (Gesteland et al., 1999). A number of facts support the RNA world concept. Nucleotide triphosphates are highly effective sources of free energy. They fulfill this function as relicts in most chemical reactions of contemporary metabolism (Stryer, 1995). Dinucleotides act as cofactors in many protein enzymes. In fact, RNA molecules can serve as enzymes (Cech, 1986) and scientists now commonly talk about ribozymes. Contemporary ribosomes translating information from RNA onto a protein structure (see Figure 1.1a) fulfill their catalytic functions due to their ribosomal RNA content rather than their protein components (Ramakrishnan and White, 1998). We have known for a number of years now about the reverse transcriptase that trans- cribes information from RNA onto DNA (Figure 1.1b). It also appears that RNA may be a primary structure and DNA a secondary one since modern organisms synthesize deoxyribonucleotides from ribonucleotides. 1.2 Machinery of prokaryotic cells The smallest present-day system thought to possess the key function of a living organism, namely reproduction, is a cell. A sharp distinction exists between simple prokaryotic cells (that do not have nuclei) and far more complex eukaryotic cells (with well defined nuclei). Evidence points to an earlier evolution of prokary- otic cells. Eukaryotic cells are believed to have resulted from mergers of two or more specialized prokaryotic cells. Unfortunately, little is known about the origins of prokaryotic cells. The scenario below is only an attempt to describe some key functional elements of the apparatus possessed by all prokaryotic cells and is not a serious effort to reconstruct the history of life on Earth. The world of competing RNA molecules must have eventually reached a point where a dearth of the only building materials, nucleotides triphosphate, was cre- ated. Molecules that could obtain adequate supplies of building materials gained an evolutionary advantage but they needed containers to carry their supplies and pro- tect them from the environment. In the liquid phase, such containers were formed spontaneously from phospholipids. The phospholipid molecules are amphiphilic — one part is hydrophilic (attracted to water) and the other is hydrophobic (repelled by water). See Section 2.4 for more details on this aspect. As a result of movement of the hydrophobic part away from water and movement of the hydrophilic part toward water, an unbounded lipid bilayer or a three-dimensional vesicle is formed (Figure 1.2). Since phospholipid vesicles can join to construct bigger structures from several small ones, they are important to the RNA molecules that can divide and compete for food. Merging into bigger vesicles can be advantageous in foraging for food. Division into small vesicles can be seen as a type of reproduction. The phospholipid vesicle was not a complete answer to the problem because it required a way to selectively infuse nucleotides into its interior. Employing new types of biomolecules — amino acids, of which some were hydrophilic and some hydrophobic, solved that problem. Their linear polymers are called peptides and long peptides give rise to proteins. Proteins possess three-dimensional structures © 2003 by CRC Press LLC4 Introduction to Molecular Biophysics FIGURE 1.2 In a water environment, amphiphilic molecules composed of hydrophilic (shaded) and hydrophobic (white) parts organize spontaneously into bilayers closed into three-dimensional vesicles. Protein, a linear polymer of appropri- atelyorderedhydrophilic(shadedcircles)andhydrophobic(whitecircles)amino acids, forms a structure that spontaneously builds into the bilayer and allows selectivelychosenmolecules,e.g.,nucleotidetriphosphates,topassintothelipid interior. whose hydrophobicity depends on the order in which amino acid segments appear in a linear sequence. Such proteins may spontaneously embed themselves in a lipid bilayer and play the roles of selective ion channels (see Figure 1.2). The first stage in the development of a prokaryotic cell was probably the enclo- sure of RNA molecules into phospholipid vesicles equipped with protein channels that enabled selective transfer of triphosphate nucleotides into the interior region (Figure 1.3a). The second stage must have been the perfection of these channels and a link between their structures and the information contained in the RNA molecules. Selective successes may have been scored by RNA molecules that could translate some of the information contained in the RNA base sequence into an amino acid sequence of an ion channel protein in order to synthesize it. This was the way to distinguish the so-called mRNA (messenger RNA) from tRNA (transfer RNA) and rRNA (ribosomal RNA). While mRNA carries information about the amino acid sequences in proteins, tRNA connects amino acids with their corresponding triple base sets. rRNA is a prototype of a ribosome, a catalytic RNA molecule that can synthesize amino acids transported to it by molecules of tRNA into proteins. These amino acids had to be first recognized by triples of bases along the mRNA (Figure 1.3b). The analysis of the nucleotide sequences in tRNA and rRNA of various origins indicates that they are very similar and very archaic. The genetic code based on sequences of triples is equally universal and archaic. Contemporary investigations of prokaryotic and eukaryotic ribosomes provided solid evidence that the main catalytic role is played by rRNA and not the proteins contained within the ribosomes. © 2003 by CRC Press LLCOrigins and Evolution of Life 5 (f) (a) (c) (d) (e) (b) glycose DNA + ADP + P NAD pyruvate + ATP + NAD NAD NTP transcriptase tRNA mRNA RNA pyruvate NADH CO 2 + + + H H + rRNA H NADH replicase + + H ATP protein mRNA ATP NTP ADP+P + i lactate + H ADP+P + i H FIGURE 1.3 Developmentoftheprokaryoticcellmachinery. (a)Theself-replicatingRNAmoleculewithasupplyofnucleotidetriphosphates (NTP)isenclosedinavesicleboundedbyalipidbilayerwithbuilt-inproteinchannelsthatallowselectivepassageofnucleotide triphosphates. (b) In an RNA chain, a distinction is made between mRNA and various types of tRNA and rRNA. rRNA is a prototype of a ribosome that can synthesize proteins based on the information encoded in mRNA. Proteins produced this way are more selective membrane channels and effective enzymes that can catalyze many useful biochemical processes. (c) Double- stranded DNA replaces RNA as an information carrier. Protein replicases double this information during division and protein transcriptases transfer it onto mRNA. (d) Protein enzymes appear to be able to catalyze lactose fermentation of sugars as a resultofwhichthepoolofhigh-energynucleotide(mainlytriphosphatesATP)canbereplenishedusinglowenergydiphosphates + (mainlyADP).Theamountofoxidizer(hydrogenacceptor)NAD remainsconstant;thecellinteriorbecomesacidic. (e)Proton + pumps can pump H ions into the cell exterior via ATP hydrolysis. (f) Other proton pumps use hydrogen obtained from the decompositionofsugarsthroughpyruvateasfuel. Duetothepresenceofawallorasecondcellmembrane,pumped-outprotons can return to the cell interior through the pumps of the first type that act in reverse to reconstruct ATP from ADP. Membrane phosphorylation becomes the basic mechanism of bioenergetics in all modern living organisms. © 2003 by CRC Press LLC6 Introduction to Molecular Biophysics Proteins have much better catalytic properties than RNA. A key property is their high specificity vis a vis the substrate. They soon (in the form of polymerases) replaced RNA in the process of self-replication. It was already possible on the RNA template to replicate sister RNA and DNA. DNA spontaneously forms a structure composed of two complementary strands (a double helix). The helix is a much more stable information carrier than RNA. This principle led to the current method of transferring genetic information (see Figure 1.3c). Genetic information is stored in double-stranded DNA. Protein replicases duplicate this information in the process of cell division. If necessary, protein transcriptases transcribe this information onto mRNA, which is used during the process of translation (partly ribozymatic and partly enzymatic) as a template to produce proteins. The transfer of information in the reverse direction from RNA to DNA via reverse transcriptases is a fossil remnant that has been preserved in modern retroviruses. Protein enzymes can perform useful tasks. They can produce much-needed triphosphate nucleotide building materials and recycle them from used diphosphates and inorganic orthophosphate, using saccharides as a source of free energy. Figure 1.4 illustrates the main metabolic pathways of energy and matter process- ing that are common to contemporary bacteria (prokaryotes) and animals and plants (eukaryotes). The central point at which many of these metabolic pathways converge is pyruvate. It is easy to see the vertical path of glycolysis, the reduction of the most common monosaccharide, glucose, to pyruvate. It is equally easy to see the circular cycle of the citric acid that is connected with pyruvate through one or more reactions. The archaic origins of the main metabolic pathways are evident in their universal- ity (from bacteria to man) and in many of the reactions of nucleotide triphosphates, mainly ATP (adenosine triphosphate). Reactions connected with the hydrolysis of ATP to ADP (adenosine diphosphate) are indicated in Figure 1.4 by P’s at the starts of the reactions. Reactions linked to the synthesis of ADP and an orthophosphate group into ATP (phosphorylation) are indicated by P’s at the ends of reactions. − The transition from glucose, C H O , to pyruvate, CH -CO-COO , is an oxi- 6 12 6 3 + dation reaction that takes hydrogen atoms from glucose molecules. NAD (nico- tinamide adenine dinucleotide) is a universal oxidant (an acceptor of hydrogen, i.e., simultaneously an electron and a proton). This process is also a relict of the RNA + world. The acceptance by NAD of two hydrogen atoms is shown in Figure 1.5 by the H at the end of each reaction. An overall balance of the glycolysis reaction or oxidation of glucose to a pyruvate takes the form: + C H O +2NAD + 2 ADP + 2P 6 12 6 i − + →2CH −CO−COO + 2 NADH +2H +2ATP +2H O. (1.1) 3 2 + Two molecules of NAD are reduced by four atoms of hydrogen: + − + + C H O +2NAD →2CH −CO−COO +2H + 2 NADH +2H (1.2) 6 12 6 3 (Two protons are obtained from the dissociation of pyruvic acid into a pyruvate anion, + whereas two other protons transfer the original positive charge of NAD ) and two molecules of ADP are phosphorylated to ATP according to the equation: + ADP + P + H → ATP + H O. (1.3) i 2 © 2003 by CRC Press LLCOrigins and Evolution of Life 7 starch, glycogen P H H P P nucleotides H phospholipids H P H P H H P H amino acids steroids H P P H H H porphyrins P H FIGURE 1.4 An outline of the main metabolic pathways. Substrates are represented by black dots; reversible or practically irreversible reactions catalyzed by specific enzymes are represented by arrows. In a neutral water environment, ATP is present as an ion with four negative charges, ADP with three negative charges, and an orthophosphate P with two. i The primitive prokaryotic cells were properly equipped with the machinery of pro- tein membrane channels able to select specific components from their environment. They also had protein enzymes to catalyze appropriate reactions. These cells became able to replenish their pools of nucleotide triphosphates at the expense of organic + compounds of a fourth type — saccharides (see Figure 1.3d). The NAD oxidant was recovered in the process of fermentation of a pyruvate into a lactate: − + − + CH −CO−COO + NADH + H → CH −CHOH−COO + NAD . (1.4) 3 3 This reaction is also used by modern eukaryotic organisms whenever they must rapidly obtain ATP under conditions of limited oxygen supply. The lactic fermentation process that accompanies phosphorylation of ADP to ATP with the use of sugar as a substrate has several drawbacks. In addition to its low © 2003 by CRC Press LLC8 Introduction to Molecular Biophysics (a) (b) + + 3H 2H + + ATP H + NADH 2H + interior NAD ADP + P interior − 2e Q − 2e - NO 3 + + - 3H 2H NO + H O 22 + 2H FIGURE 1.5 + Proton pumps transport free proton H across the membrane from the cell interior to its exterior at the expense of the following chemical reactions: hydrolysisofATPintoADPandaninorganicorthophosphate(a)oroxygenation ofhydrogenreleasedinthedecompositionofglucosetoCO andtransportation 2 + by NAD . (b) A derivative of quinone Q is an intermediary in hydrogen trans- port. Themoleculeissolubleinsidethemembraneandoxidationisaccomplished − − through NO reduced to NO . If pumps of both types are located in the same 3 2 membrane, the first protons passing in the reverse direction can phosphorylate ADP to ATP. efficiency (unused lactate), it leads to increased acidity of the cells. While sugars are neutral (pH near 7), lactate is a product of dissociation of lactic acid and in the + process of breakdown of sugars, a free proton H is released. The lowering of pH results in a significant slowdown or even stoppage of the glycolysis reaction. For the decomposition of sugars to be effectively used in the production of ATP, a cell must find a different mechanism of fermentation whose product has a pH near + 7 or whose proton H can be expelled outside the cell. In yeast, a new type of fermentation consists of the reduction of pyruvate to ethanol with a release of carbon dioxide in the process: − + + CH −CO−COO + NADH +2H → C H −OH + CO + NAD . (1.5) 3 2 5 2 Before this mechanism had been adopted, a proton pump was discovered utilizing the hydrolysis of ATP as a source of energy (Figure 1.3e). During the production of one + molecule of ATP, one hydrated proton H is released inside the cell. The hydrolysis of one molecule of ATP results in the pumping outside the cell membrane of three + hydrated H protons (see Figure 1.5a). The process is still energetically favorable. However, from the viewpoint of ATP production, a more efficient process is further oxidation of a pyruvate to an acetate and a carbon dioxide: − + − + CH −CO−COO + H O + NAD →CH −COO + CO + NADH + H . (1.6) 3 2 3 2 + The equation above shows a reduction of one molecule of NAD by two atoms of hydrogen. Subsequently, in the citric acid cycle of Krebs (see Figure 1.4), © 2003 by CRC Press LLCOrigins and Evolution of Life 9 Gram - positive Gram - negative FIGURE 1.6 Abacterialcellisequippedwithacellwallcomposedofpeptidoglycan,acomplex protein-polysaccharide structure (shaded). It can also have a second, external membrane. TheexposedthickpeptidoglycanlayerchangesitscolorintheGram dyeing procedure. The thin peptidoglycan layer covered by the external mem- brane does not change color. Hence bacteria are categorized as Gram-positive and Gram-negative. acetate is oxidized to carbon dioxide and water. The net balance in the Krebs cycle is: − + + + CH −COO + H +2H O + 3 NAD + FAD + GDP + P + H 3 2 i + →2CO + 3 NADH +3H + FADH + GTP + H O. (1.7) 2 2 2 Acetate enters the reaction bound to a so-called co-enzyme A (CoA) as acetyl CoA. + During one turn of the Krebs cycle, a further reduction of three molecules of NAD and one molecule of FAD (flavin adenine dinucleotide) involving eight atoms of hydrogen and phosphorylation of a molecule of GDP (guanosine diphosphate) to GTP (guanosine triphosphate) takes place. To enhance clarity, we used square brackets to indicate subprocesses. Discussing the economy of the Krebs cycle makes sense only when a cell is able to + utilize fuel in the form of hydrogen bound to the NAD and FAD carriers for further phosphorylation of ADP to ATP. This became possible when a new generation of proton pumps was discovered. These pumps work as a result of the decomposition of hydrogen into a proton and an electron instead of ATP hydrolysis. These particles are further transported along a different pathway to the final hydrogen acceptor which, in the early stages of biogenesis, may have been an anion of an inorganic acid. Primitive bacterial cells were endowed with cell membranes composed of peptido- glycan, a complex protein–saccharide structure, and later developed additional cell membranes (see Figure 1.6). This facilitated accumulation of protons in the spaces outside the original cell membrane from which they could return to the cell interi- ors using the proton pump of the first type (see Figure 1.3f). This pump, working in reverse, synthesizes ATP from ADP and an orthophosphate. This very efficient © 2003 by CRC Press LLC10 Introduction to Molecular Biophysics mechanism of membrane phosphorylation is universaly utilized by all present-day living organisms. A more detailed explanation of the proton pump that utilizes the oxidation of hydrogen is shown in Figure 1.5b. In the original bacterial version, the pump is composed of two protein transmembrane complexes: a dehydrogenase of NADH − − and a reductase, for example, the one changing the nitrate NO to nitrite NO .In 3 2 the first complex, two hydrogen atoms present in the pair NADH–hydronic ion are transferred to FMN (flavin mononucleotide). Later, after two electrons are detached, the hydrogens (as protons) are transferred to the other side of the membrane. The two electrons are accepted in turn by one and then another iron–sulfur center (iron is 3+ 2+ reduced from Fe to Fe ). Subsequently, at a molecule of quinone derivative Q, they are bound to another pair of protons that reached the same site from the interior of the cell. An appropriate derivative of quinone Q is soluble inside the membrane and serves as an intermediary that ferries two hydrogen atoms between the two complexes. At the other complex, two hydrogen atoms are again split into protons and electrons. The released protons are transferred to the exterior of the membrane and the electrons and the protons from the interior of the membrane are relocated to a final acceptor site that may be a nitrate anion. Thus, the created nitrite can oxidate another reaction that can be used by another reductase: − NO → N . (1.8) 2 2 Alternatively, the nitrite can be involved with other inorganic anions such as an acid carbonate or sulfate in reactions leading to the formation of compounds with hydrogen: ammonia, methane, or sulfurated hydrogen: − − 2− NO → NH , HCO → CH , SO → H S. (1.9) 3 4 2 2 3 4 1.3 The photosynthetic revolution The Earth is energetically an open system and a substantial flux of solar radiation has reached it since the moment of its creation. Along with the rotational motion of the planet, the flux has powered the machinery that produces oceanic and atmo- spheric motions. The primary energy sources for the newly emerged life on Earth were nucleotide triphosphates and exhaustible supplies of small organic molecules such as monosaccharides. Life became energetically independent only when organ- isms learned how to harness practically inexhaustible solar energy or, more precisely, the fraction of it that reaches the surfaces of the oceans. The possibility of utilizing solar energy by living cells is linked to the use of chlorophyll as a photoreceptor (Nitschke and Rutherford, 1991). The chlorophyll molecule contains an unsaturated carbon–nitrogen porphyrin ring (see Figure 1.4) 2+ with a built-in Mg ion and phytol, a long saturated hydrophobic carbohydrate chain. The molecules of chlorophyll are easily excited in the optical range and easily transfer this excitation among each other, creating a light harvesting system in an appropriate protein matrix. The last chlorophyll molecule in such a chain can © 2003 by CRC Press LLCOrigins and Evolution of Life 11 + + 2H 2H interior RCII cytbc1 Q - 2e - 2e c H S 2 S - 2e + 4H + 2H FIGURE 1.7 A proton pump in purple bacteria utilizing solar radiation energy. In the first protein complex (type II reaction center or RC II), an electron from an excited chlorophyllmolecule(theprimarydonor)istransferredtoamoleculeofquinone derivative(Q)alongwithaprotonfromthecellinterior. Thequinonederivative moleculecarriestwohydrogenatomsformedthiswaytoanotherproteincomplex containingcytochromebc1. Hydrogenatomsinthecomplexareagainseparated. A proton is moved outside the cell while an electron reduces a molecule of the water-soluble cytochrome c, which carries it back to the primary donor. An alternative source of electrons (broken line) for sulfur purple bacteria can be a molecule of sulfurated hydrogen (H S). 2 + become an electron donor and replace the NADH + H fuel in a proton pump (see Figure 1.5b). The first organisms to avail themselves of this possibility were probably purple bacteria. Their proton pumps are two protein complexes built into the cell membrane (see Figure 1.7). In the protein complex called the type-II reaction center (RC), two electrons from the excited chlorophyll are transferred with two protons from the cell interior to a quinone derivative Q with a long carbohydrate tail. Q is soluble in the membrane. When reduced to quinol QH , it carries the two hydrogen atoms 2 inside the membrane to the next complex that contains a protein macromolecule called cytochrome bc1. The macromolecule catalyzes the electron transfer from each hydrogen atom onto another macromolecule called cytochrome c while the remaining proton moves to the extracellular medium. Cytochrome proteins contain a heme in the form of a porphyrin ring with a built-in 2+ 3+ Fe ion that may also exist in a form oxidized to Fe . Cytochrome c is a water- soluble protein that removes electrons outside the cell membrane and returns them to the reaction center. This completes the cyclical process during which two protons are carried from inside the cell to the outside. Alternative sources of electrons needed to restore the initial state of the reaction center used, for example, in sulfur purple bacteria may be the molecules of sulfurated hydrogen H S. Contrary to the oxidation 2 of NADH, oxidation of H S to pure sulfur is an endoergic reaction (consuming and 2 not providing free energy) and it cannot be used in proton pumps. The proton concentration difference on each side of the cell membrane is further used by purple bacteria to produce ATP the same way it is produced by nonpho- tosynthetic bacteria. Green bacteria found an alternative way of using solar energy © 2003 by CRC Press LLC12 Introduction to Molecular Biophysics + 2H + NADP + Fd H + NADPH interior - 2e RC I - 2e H S 2 S + 2H FIGURE 1.8 The utilization of solar energy by green sulfur bacteria. In the first protein complex(typeIreactioncenterorRCI),anelectronfromanexcitedchlorophyll molecule is transferred to a water-soluble protein molecule of ferredoxin (Fd) + which carries it to the complex of NADP (nicotinamide adenine dinucleotide phosphate) reductase. The deficit electron in the initial chlorophyll is compen- sated in the process of oxidation of sulfurated hydrogen (H S). The reduced 2 + hydrogencarrier(NADPH+H )servesasfuelintheCalvincyclesynthesizing sugar from water and carbon dioxide. (see Figure 1.8). In the protein complex called the type I reaction center, an electron from photoexcited chlorophyll is transferred to a water-soluble protein called ferre- doxin. The lack of electrons in the chlorophyll molecule is compensated uncyclically from sulfurated hydrogen decomposition. The electron carrier in ferredoxin is the iron–sulfur center composed of four Fe atoms directly and covalently bound to four S atoms. After the reduction of iron, ferredoxin carries electrons to the next protein complex where the electrons bind + to protons moving from the cell interior and reducing the molecules of NADP + (nicotinamide adenine dinucleotide phosphate) to NADPH+ H . The entire system is not really a proton pump since no net proton transport occurs across the cell membrane. The system transforms light energy into fuel energy in the molecules of NADPH + + together with hydrated protons H that carry the original charge of NADPH . This fuel is used in the synthesis of glucose from CO and H Ointhe Calvin cycle whose 2 2 overall balance equation takes the form: + 6 CO + 12 NADPH + 12 H + 18 ATP + 18 H O→C H O 2 2 6 12 6 + + +6H O + 12 NADP + 18 ADP + 18 P + 18 H . (1.10) 2 i This cycle is in a sense a reverse of the Krebs cycle. Analogously to the Krebs cycle, we used square brackets to denote summary component reactions in order to show more clearly the net reaction. ATP is also used in the Calvin cycle. After the oxidation of glucose in the same way as for nonphotosynthetic bacteria, an excess of ATP is produced. © 2003 by CRC Press LLCOrigins and Evolution of Life 13 + 2H + + + NADP 2H 2H + Fd H + NADPH stroma - 2e PSII PSI cytbf Q - 2e - 2e - H O 2e PC 2 1 O - 2 2 + 4H + 2H FIGURE 1.9 Aprotonpumpusingsolarenergyincyanobacteriacanbethoughtofasacombi- nationoftheprotonpumpinpurplebacteria(typeIIreactioncenter,nowcalled photosystem II or PS II) and the photosynthetic system of green bacteria (type I reaction center, now called photosystem I or PS I). The coupling of the two systems is done by a water-soluble molecule of plastocyanin (PC) with a copper ionservingasanelectroncarrier. Thefinalelectrondonoriswaterwhich,after donating electrons and protons, becomes molecular O . The proton concentra- 2 tion difference between the two sides of the cell membrane is used to produce + ATP via H ATPase (see Figure 1.5a) working in the reverse direction. In prin- ciple,thephotosyntheticsystemsinthetylakoidmembranesofchloroplaststhat are organelles of eukaryotic plant cells are identical structures. Combining the two methods of using solar energy offers the optimal solution. In cyanobacteria, cytochrome c1 was replaced by cytochrome f , whereas cytochrome c was replaced by plastocyanin (PC) and used as an electron carrier between type II and type I reaction centers. The centers are now known as photosystem II (PS II) and photosystem I (PS I), respectively (Figure 1.9). The electron carrier in plastocyanin is 2+ + the Cu copper ion which is reducible to Cu and directly bound via four covalent bonds to four amino acids: cysteine, methionine, and two histidines. The greatest breakthrough resulted not from the combination of the two photo- systems, but from the utilization of water as the final electron donor (and a proton donor, hence a hydrogen donor). The dissociation of hydrogen atoms from a water molecule turned it into a highly reactive molecular O gas that was toxic to the early 2 2+ biological environment. Initially, it oxidized only Fe ions that were soluble in great quantities in contemporary ocean water. As a result of this oxidation, poorly soluble 3+ Fe ions were formed. They sedimented, giving rise to modern iron ore deposits. The increased production of sugars from CO and H O reduced ocean acidity and 2 2 − 2− caused a transformation of acidic anions of HCO into neutral CO ions. The 3 3 2− 2+ CO reacted with the Ca ions initially present in high concentrations, leading 3 to sedimentation of insoluble calcium carbonate CaCO . The membranes of cyano- 3 bacteria captured the calcium carbonate and produced a paleobiological record of these processes in the form of fossils called stromatolites. The formation of calcified stromatolites depleted the atmosphere from CO . When 2 a deficit of compounds capable of further oxidation occurred, molecular O started to 2 be released into the atmosphere. Along with molecular N formed by the reduction of 2 nitrates, the O brought about the contemporary oxygen–nitrogen based atmosphere 2 © 2003 by CRC Press LLC14 Introduction to Molecular Biophysics + 2H + + H + NADH + 4H 2H + NAD interior cytbc1 cytaa3 Q - 2e - 2e 1 c -O - 2 2 2e + 2H H O 2 + 4H + 2H FIGURE 1.10 Protein pump of heterotrophic aerobic bacteria. Electrons from the fuel in + the form NADH + H (produced in glycolysis and in the Krebs cycle) are transferred via quinone (Q) to the protein complex with cytochrome bc1 and then via cytochrome c to the complex with cytochrome aa3. The final electron acceptor is molecular O . During the transfer of two electrons along the mem- 2 brane,eightprotonsarepumpedacrossit. Theprotonconcentrationdifference + betweenthetwosidesofthemembraneisusedtoproduceATPbyH ATPase(see Figure1.5a)workinginreverse. Thisisinprincipleidenticaltothemechanismof oxidativephosphorylationinthemitochondrialmembrane,whichisanorganelle present in all eukaryotic cells. containing only trace quantities of carbon dioxide. Life had to develop in a toxic oxygen environment from that point onward. The problem was solved by the mech- anism of oxidative phosphorylation used by modern aerobic bacteria and all higher organisms. A proton pump that used inorganic anions as final electron acceptors (see Figure 1.5b) was replaced by a pump in which the final electron acceptor is molecular oxygen (see Figure 1.10). The cytochrome bc1 transfers electrons from quinone Q to water-soluble cytochrome c, a mechanism utilized earlier by purple bacteria (see Figure 1.7). + The source of electrons transferred to the quinone can be the NADH + H gen- erated by glycolysis and in the Krebs cycle or, directly, FADH (reduced flavin ade- 2 nine dinucleotide) produced in one stage of the Krebs cycle (oxidation of succinate to fumarate). Electrons can also come from an inorganic source (chemotrophy). For example, nitrifying bacteria can oxidize ammonia to nitrate using molecular oxygen: − − NH → NO → NO (1.11) 3 2 3. Nature demonstrates here, as it has many times, its ability to use environmental pollution to its advantage. It will be interesting to see, for example, what use it finds for the countless tons of plastic bottles deposited in modern garbage dumps. © 2003 by CRC Press LLCOrigins and Evolution of Life 15 1.4 Origins of diploidal eukaryotic cells In its 19th century interpretation, Darwin’s theory of natural selection favoring survival of the fittest could be readily associated with the contemporary struggle for survival in the early capitalist economy of that time. Both endeavors were ruled by the law of the jungle that became anathema to the ideological doctrines of many totalitarian regimes on the 20th century political landscape. The great biologist, Lynn Margulis (1998), emphasized the fact that survival can be accomplished through strug- gle or through peaceful coexistence (symbiosis is the biological term). Many clues support the significance of symbiosis in the formation of modern eukaryotic cells. Figure 1.11 illustrates a simplified phylogenetic tree of living organisms. It shows a clear division between archaic bacteria (Archaebacteria) and true bacteria (Eubacteria) that may have emerged in the earliest periods of life on Earth. The history of subsequent differentiation of prokaryotic organisms within these two groups, however, is not all that clear. The modern phylogenetic tree is based on differences in DNA sequences coding the same functional enzymes or ribozymes. The more differences found in the DNA sequences, the earlier the two branches of the com- pared species must have divided. The results obtained by comparing, for example, ribosomal RNA with the genes of the proteins in the photosynthetic chain differed greatly and led to very dissimilar reconstructions of the history of the evolution of photosynthesis (Doolittle, 1999; Xiong et al., 2000). The reason for this ambiguity is the lateral gene transfer process by which genes are borrowed by one organism from another. Branches can split away and merge over time. Therefore, the phylogenetic tree of Eubacteria shown in Figure 1.11 must be viewed with caution, especially since only the kingdoms essential to our discussion are depicted. Gram-positive bacteria have only single external membranes and are potentially sensitive to antibiotics. Fortunately, the group includes most of the pathogenic bacteria. Spirochetes have developed mechanisms of internal motion for entire cells. Photosynthetic purple bacteria with type II reaction centers can be sulfuric or non- sulfuric. They must be evolutionarily close to aerobic bacteria because they utilize the same mechanism of reduction of cytochrome c through the protein complex with cytochrome bc1 (see Figures 1.6 through 1.10). Biologists do not distinguish king- doms of bacteria by this characteristiic. Most aerobic bacteria, including the common Escherichia coli, can survive in oxygen-deprived conditions. Green bacteria and cyanobacteria have in common the mechanism of sugar photosynthesis via the use of type I reaction centers. Lateral gene transfer can be accomplished when several simple eukaryotic cells merge into one supercell. According to Margulis, this is how eukaryotic cells first formed. Most probably, a thermophilic bacterium with a stable genomic organization whose DNA was protected by proteinaceous histones that combined to form a chro- matin prototype entered into a symbiotic arrangement with a spirochete containing a motile apparatus formed from microtubules (see Figure 1.11). This combination gave rise to a mitotic mechanism of cell division (Solomon et al., 1993). Chromatin with a doubled amount of genetic material organized itself after replication into © 2003 by CRC Press LLC16 Introduction to Molecular Biophysics halophiles methanogens thermophiles fungi nucleus animals protista plants centrioles mitochondria chloroplasts Gram-positive spirochets purple aerobic green cyanobacteria FIGURE 1.11 Simplified and somewhat hypothetical phylogenetic tree of living organisms. The earliest are the two groups of prokaryotic organisms, Archaebacteria and Eubacteria. As a result of the merger of prokaryotic cells with different pro- perties, eukaryotic cells (Eucarya) were formed. Eucarya further evolved in undifferentiated forms as single cell organisms (protista kingdom) or differen- tiated into multicellular organisms (the kingdoms of heterotrophic fungi with multinuclear cells, heterotrophic animals, and phototrophic plants). chromosomes pulled in opposite directions by a karyokinetic spindle formed from centrioles by self-assembling microtubules that consumed GTP as fuel. In the next stage, cells with nuclei that contained chromatin assimilated several oxygen bacteria (see Figure 1.11). The oxygen bacteria were transformed into mitochondria, the power plants of cells that synthesized ATP via the phosphoryla- tive oxidation mechanism shown in Figure 1.10. The formed Eucarya cells continued evolving (Figure 1.11) in undifferentiated forms as single-cell organisms (the protista kingdom) or in differentiated forms as multicellular organisms (fungi and animal king- doms). All the original organisms were heterotrophs. The assimilation of prokaryotic cells of cyanobacteria as chloroplasts led to the formation of phototrophic single-cell organisms and multicellular plants. So far, we have only discussed the symbiosis of prokaryotic cells. An encounter of two organisms belonging to the same species will lead to cannibalism or © 2003 by CRC Press LLCOrigins and Evolution of Life 17 symbiosis. According to Margulis, symbiotic encounters led to the emergence of sex. A symbiotic cell becomes diploidal, i.e., contains two slightly different copies of the same genome. Obviously, reproductive cells nurtured by a parent organism before entering into new symbiotic arrangements are haploidal and contain only one copy of genetic material. A reduction of the genetic information took place in the process of generating reproductive cells when meiotic division replaced mitotic division. The evolutionary advantage of sexual reproduction is due to the crossing-over of maternal and paternal genes in meiotic division. As a result, the genetic material undergoes a much faster variability compared to random point mutations and such recombinations are seldom lethal. Figure 1.12 illustrates a eukaryotic cell composed of a system of lipid membranes that confine its various organelles (Solomon et al., 1993): the nucleus, mitochondria, smooth and rough endoplasmic reticulum, Golgi apparatus, and lysosomes. The centrioles organize the motile system. The illustration corresponds to an animal cell. Plant cells (see Figure 1.13) contain additional cell walls, vacuoles that store water, and various additional substances in the form of grains and chloroplasts — large organelles consisting of three layers of membranes that facilitate photosynthesis. The internal flattened chloroplast bubbles are called thylakoids; they can be viewed as removed mitochondrial crista (combs). Three types of organelles of eukaryotic organisms contain their own genetic material, which is different from that of the nucleus. As mentioned earlier, eukary- otic systems emerged in the process of evolution due to the assimilation of previously developed prokaryotic cells. These are mitochondria that originated from oxygen bacteria, the centrioles originating from spirochetes and chloroplasts formed from cyanobacteria. Figure 1.4 depicts the broad scheme of metabolism; it shows only the most impor- tant biochemical reactions. We now know that almost 100 times as many reactions take place in a cell (Stryer, 1995). Substrates are denoted by dots. A unique enzyme catalyzes each reaction (represented by a single or bidirectional arrow). Worth men- tioning are vertical chains of sugar transformations (left side), fatty acids (right side), and a characteristic closed Krebs cycle. The diagram also shows how connections are made with more complex reaction systems transforming other important bio-organic compounds such as nucleotides, amino acids, sterols, and porphyrins. Specific reactions occur in each cell compartment. Biopolymer hydrolysis takes place in lysosomes. Krebs reaction cycles and fatty acid and amino acid degrada- tion can be found in the mitochondrial matrix. Oxidative phosphorylation reactions proceed on internal membranes of mitochondrial. Protein synthesis takes place on membranes of the rough endoplasmic reticulum and lipid synthesis can be observed on membranes of the smooth endoplasmic reticulum. Compound sugar synthesis occurs in the interior of the Golgi apparatus. After merging with membrane proteins, sugars synthesized in the Golgi apparatus can be transported outside cells in a process called exocytosis. The external cytoplas- mic membrane armed with such glycoproteins recognizes and selectively transports substances from the external environments of the cells. Transformations of simple sugars, amino acids, and mononucleotides take place between various organelles in © 2003 by CRC Press LLC18 Introduction to Molecular Biophysics cilium (short) or flagellum (long) centrioles smooth endoplasmic reticulum nucleus rough endoplasmic reticulum Golgi complex mitochondrium egzosome lyzosome endosome FIGURE 1.12 Compartments of a eukaryotic cell. Solid lines represent membranes formed from two phospholipid bilayers and the neighboring compartments differ in degrees of shading. © 2003 by CRC Press LLCOrigins and Evolution of Life 19 granule vacuole thylakoid chloroplast cell wall FIGURE 1.13 Additional organelles of a plant cell. the cellular interior. The region is filled simultaneously with supramolecular struc- tures of the protein cytoskeleton that provide the cell with motile machinery. 1.5 Summary: further stages of evolution We will summarize here the most important stages in the evolution of life on Earth from the Big Bombardment to the achievement of supracellular levels of organization (Solomon et al., 1993). Some of the elements of the puzzle are well known; they left visible traces that were precisely dated. Some elements are somewhat hypothetical and have not yet been backed by solid discoveries and analyses. They are indicated by question marks. © 2003 by CRC Press LLC

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