Evolution was Chemically Constrained

J. theor. Biol. (2003) 220, 323–343 doi:10.1006/jtbi.2003.3152, available online at http://www.idealibrary.com on

Evolution was Chemically Constrained R. J .P. Williamsnz and J. J. R. FrauŁsto Da Silvaw wInorganic Chemistry Laboratory, University of Oxford, South Parks Road, Oxford OX1 3QR, U.K. and zDepartment of Chemistry, Instituto Superior Te´cnico, Lisbon, Portugal (Received on 21 March 2002, Accepted in revised form on 22 August 2002)

The objective of this paper is to present a systems view of the major features of biological evolution based upon changes in internal chemistry and uses of cellular space, both of which it will be stated were dependent on the changing chemical environment. The account concerns the major developments from prokaryotes to eukaryotes, to multi-cellular organisms, to animals with nervous systems and a brain, and finally to human beings and their uses of chemical elements in space outside themselves. It will be stated that the changes were in an inevitable progression, and were not just due to blind chance, so that ‘‘random searching’’ by a coded system to give species had a fixed overall route. The chemical sequence is from a reducing to an ever-increasingly oxidizing environment, while organisms retained reduced chemicals. The process was furthered recently by human beings who have also increased the range of reduced products trapped on Earth in novel forms. All the developments are brought about from the nature of the chemicals which organisms accumulate using the environment and its changes. The relationship to the manner in which particular species (gene sequences) were coincidentally changed, the molecular view of evolution, is left for additional examination. There is a further issue in that the changes of the chemistry of the environment developed largely at equilibrium due to the relatively fast reactions there of the available inorganic chemicals. Inside cells, some of these same chemicals also came to equilibrium within compounds. All such equilibria reduced the variance (degrees of freedom) of the total environmental/biological system and its possible development. However, the more sophisticated organic chemistry, almost totally inside cells until humans evolved, is kinetically controlled and limited by the demands of cellular reduction necessary to produce essential chemicals and by the availability of certain elements and energy. Hence the variability of reductive cellular organic chemistry and its limitations in cells have to be considered separately. While as a whole they drive the oxidation of the environment, they also allow speciation within the major changes of organisms. Human beings have introduced recently new, virtually irreversible, inorganic and organic chemistry in the environment, much of it new modes of irreversible storage of reduced chemicals, and this is, we state, the last possible step of chemical evolution. We must attempt to evaluate its effect on organisms generally. It must be clear that all the changes and the original life forms are dependent upon energy as well as material capture and flow. We shall have to consider in which forms energy was n Corresponding author. Tel.: +44-1865-270000; fax: +44-1865-272690. E-mail address: [email protected] (R.J.P. Williams).

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available over the period of evolution, how it was usefully transformed, and the ways in which its sources changed. r 2003 Elsevier Science Ltd. All rights reserved.

Introduction We can consider biological chemical systems from two points of view. In the first, a top-down approach, properties of the whole system often related to thermodynamic constants dominate, while in the second, a bottom-up approach, properties of extracted isolated molecules are examined initially (Van de Vijver, 1999). The second is the general method of molecular biology (Woese, 1998; Caporale, 1999). In the top-down approach consideration of variance, degrees of freedom, of systems proceeds in part from analysis of components. They are limited by equilibria and depend on the number of nonexchangeable elements in different molecules which can then be used in an examination of the variable, that is composition. Notice that the concentrations of the components are important in these variables. A typical study of the composition variable examines also interactions, organization and order between components, and the effect of other physical variables such as temperature and pressure on the system. The introduction of separate spatial compartments further affects the number of variables and where there are exchange equilibria between them the compartments define the phases present for each component. Additional phases limit variance but additional non-equilibrated compartments increase them. In biological systems compartments are not in equilibrium and therefore they increase variables. In chemical systems, mostly inorganic systems, the equilibrium approach is typically presented in phase diagrams in which variables are plotted against one another. Such properties as melting and boiling points, on appearance of new phases, are sometimes called emergent properties of the systems. (Usually such properties are not found for individual molecules, but see polymers (Williams & Frau´sto da Silva, 1999)). In the bottom-up approach, individual molecules, different from thermodynamic components (Williams & Frau´sto da Silva, 1996), are

separated and examined. The sequences of individual molecules relate to species differences and they are the variances of evolution. These molecules such as DNA, RNA and proteins (enzymes) are examined in turn for their chemical and structural properties and their relationships (Woese, 1998). In this reductionist analysis it is hoped that the study of these individual units, not usually including concentration or dynamic properties, will yield enough information for us to be able to describe organisms and their relationships. For example the examination of the sequences of the genome, DNA by itself or of RNA, falls into this class in an effort to define evolutionary trees. The examination of the protein complement of a cell, the proteome, by separation techniques (Blackstock & Mann, 2000) is somewhat different in that amounts of isolated proteins are measured, but if this is done by the study of a whole cell, it is still a bottom-up approach since it cannot discover dynamics, concentrations or positions of components in compartments. In this bottom-up molecular approach, there is no reason for evolution except competition for resources. In this article we shall use systems analysis in a simplified manner to examine features of the evolution of organisms (Williams & Frau´sto da Silva, 1999). This means that we study the introduction of extra components, by reference to element chemistry and compartments not changes internal to large molecules. We are not then concerned with the origin of life, or with the evolution of individual species. We shall state that there is an inevitable and therefore logical chemical sequence to evolution. Our conclusion parallels that of Jorgensen et al. (2000) and Corning (2002), who have looked at ecosystem emergence in general thermodynamic terms. We start from the assumption that life began in a cell, a single compartment linked to the environment, though not in equilibrium with it, since its essence was the use of energy as well as material flow from the environment through

EVOLUTION WAS CHEMICALLY CONSTRAINED

a limiting membrane. As in any such open chemical system, this chemical compartment must therefore be considered together with its adjacent external environmental compartments. In the case of the most primitive organisms these were the atmosphere and the waters around such cells, the dominant water being the sea. It follows that we must analyse the original sea and atmosphere and see them as limiting life. We show below that chemicals in the sea and atmosphere were usually in chemical and physical equilibrium. We then enquire to what extent have the changes with time of the chemical components or the physical conditions, temperature and pressure, in these environments forced the evolution of organisms? It is reasonable to assume that organisms, the sea and the atmosphere have existed at roughly constant temperature, 300750 K, and total pressure, 1–5 atm for 4.5  109 years. Since these variations are small compared with the changes in chemical components during this time, see Table 1 (and Fig. 4), it is the composition, or better collectively the components, which is a major variable of the environment and of organisms together with changes in the number and kinds of compartments. We can ask further whether these two changes are of necessity linked in evolution. This is tantamount to saying that we look for restrictions on the chemistry of systems which can give rise to evolution not through random coding (which in itself has no limitations) but by certain rules, unavoidable sequences of states and combinations of chemical elements, some of which may be in equilibria. Therefore, the approach to the chemicals involved has four parts: first, what was the nature of the initial chemistry of organisms? Second, what compositional limitations and equilibria of external environmental components have there been which we can relate to the chemical elements and which changed with time? Third, how did these limitations affect internal chemical constraints in cells with time including any further equilibria? (Note that equilibrium considerations do not apply to the general synthesis of organic molecules but they do often limit the interactions between molecules and elements in a cell.) The fourth question relates to the variance (degrees of freedom) which was introduced when

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Table 1 Available free concentrations in the sea as they changed with time Metal ion

Original conditions (M)

Aerobic conditions (M)

Na+ K+ Mg2+ Ca2+ Mn2+ Fe Co2+ Ni2+ Cu Zn2+ Mo W H+ H2S

4101 B102 B102 B103 B106 B107(FeII) o109 o109 o1020 (very low), CuI o1012 (low) o1010 ½MoS2 4 ; Mo(OH)6] B109 ½WS2 4  PH low (6.5?) 102

HPO2 4

o103

4101 B102 4102 B103 B108 B1019 (FeIII) B(109) o109 o1010, CuII o108 108 ðMoO2 4 Þ 109 ðWO2 4 Þ pH 7.6–8.2 (sea) Low [ SO2 4 (102)] 3 o10

organisms were contained not in one but in several (linked) compartments. In what ways were the types of compartment which cells developed necessitated by the need for new organized activity? Finally we may look at human chemistry and its influence on the variables. As stated, organisms require energy from outside themselves and therefore we shall have to ask also about the energy available to them from the environment, that is the atmosphere, mineral deposits, the sun and the sea (Baltscheffsky, 1996). The energy input can be looked upon as a further variable of nonequilibrium thermodynamics. If the supply of energy changes over time, then evolution of life will be sensitive to that change. This means that the energy available to organisms can only be looked upon as being in a given steady state over particular periods of time. There has been little change in energy available from primitive resources, the sun and Earth’s interior, but there has been a steady and considerable progression in energy sources through environmental chemical changes over 4.5  109 years, Table 2. The contents of the atmosphere and the sea have become energized slowly relative to their early state as we shall see. This has happened through disproportionation and separation of oxidation/

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Table 2 Sources of energy Period (years ago) 9

Initial (4.5  10 )

Energy sources (a) The sun (b) Basic unstable chemicals in the crust (c) Chemicals stored at high temperature in the core

After say 1 billion years (3.5  109) (a) As above (b) Some oxidized materials, some SO2 4 ; very little O2, H2O2 After say 2 billion years (2.5  109) (a) As above (b) Further oxidized materials, modest O2, H2O2 After say 3 billion years (1.5  109) (a) As above (b) Further oxidized materials, almost 50% final O2 pressure Today

(a) As above (b) Man’s fuels (c) Atomic energy

reduction states of certain elements with retention of the more reduced chemicals in organisms outside the environment. We shall therefore choose to look upon successive periods of say 500  106 years as of fixed energy and chemical supply. Many changes of energy supply have depended on certain equilibria in the sea and atmosphere so that many of the chemicals available to organisms for energy and synthesis changed together. Now when we examine systems we find that the occurrence of components at equilibrium is due to their thermodynamic stability measured at various compositions which refers to their minimum Gibb’s free energy, when many of their phases may occur together, Fig. 1. We need a corresponding approach to living non-equilibrium systems, which we shall take to be their survival strength when again species can exist in balance opposite a given composition. Survival is also a product of reproduction rate and the time a single organism exists. Generally reproduction rate diminishes with chemical complexity in the sequence from prokaryotes to humans which we are describing and hence lifespan had to increase if such evolution was to occur. Lifespan is a function of such factors as ability to capture food (elements) and energy and to manage and to protect the chemical activity in the organism. What was it that allowed or we say forced complexity and survival to increase? We

Fig. 1. The parallel between diagrams of thermodynamic stability of phases plotted against composition and that of kinetic competence or survival in a flowing system such as an organism also plotted against composition. Thermodynamic stability increases with DG, reduction of free energy. Chemical compositions intermediate between maxima species to left and right close to maxima exist in balance.

EVOLUTION WAS CHEMICALLY CONSTRAINED

wish to show that once life had started, certain chemical changes in the environment forced the changes to gain survival strength as well as to generate different kinds of organisms. We may then refer to diagrams as shown in Fig. 1. Not surprisingly we find balanced survival of all kinds of systems from simple fast-reproducing prokaryotes to complicated slow-reproducing, better-protected, higher eukaryotes, which can be chemically distinguished. Since they share a common environment and because they all use much of the same internal cytoplasmic chemistry (see below), the survival of the whole set of organisms is increased if the fast-reproducing simple species provide the complex species with essential basic, often synthesized, components while the complex species provide protection for the simple species. Cooperative linked activity of a large number of different kinds of organism from prokaryotes to the most advanced eukaryotes then has a greater survival potential than competitive activity between complex and simple organisms. (We observe generally greater competition between very similar organisms than between very different ones). In other words, an ecosystem has optimal survival strength. Note the similarity to the coincidental occurrence of phases, Fig. 1 (top). Ultimately it is the overall effort of the ecosystem to optimalize in organisms the retention of free energy (energy coming initially from the sun) which drives the whole of this chemistry towards an evolutionary limit, see Note at the end of this article. As stated earlier when we discuss evolution here we shall not expect to distinguish species differences such as those between different animals (see Woese, 1998). For example the whole of the last 500 million years, until human beings appeared, is treated as one, the next to last, period of evolution in which there were no major chemical or physical environmental changes. In this period there has been virtually no change of the oxygen content of the atmosphere and hence no change in the equilibrated environment. Our task is to explain the survival advantages and disadvantages which accrued up to about this time from major changes in available chemicals, and in energy sources. We shall say these changes led inevitably to the evolutionary progression along two series: one,

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of the utilization of chemicals to provide both new components and energy sources within a fixed number of compartments and two of increasing numbers of compartments in organisms. This leads to the observed sequence: single prokaryote cells, single eukaryote cells, multicellular eukaryote organisms and their further development in animals within a total ecosystem. This progression is clearly one of increasing number of non-equilibrated compartments, not phases in equilibrium, and therefore of the numbers of degrees of freedom and organizational complexity. Yet since multiple compartments belong to single organisms, compartments must be integrated in some way, that is their variables have to be constrained. We shall find that compartments only increased in number alongside interconnected communication also utilizing the chemical changes. The relationship of the components in these new compartments and their inter-communication are then a major part of evolution which again we shall relate to the progressive changes of availability of elements and energy-containing chemicals. The success of the progression required the ability to reproduce which was of course dependent on a code and the ability of it to adapt to the inevitable environmental changes. This may have occurred by selection under the pressure of the environment but that is not our concern and we shall not seek a link to a code, genes. We shall only consider human chemical intervention in the last section of this article. It may be helpful to the reader to recognize immediately that there are common analytical features of the central compartment, the cytoplasm, of all cells present through all evolution so that we search first for the reasons for this particular initial composition and any underlying equilibria which restrict its variety de Duve, (1991). This single central compartment was of necessity in direct supply communication with the primitive environment, to which we turn shortly. It is the chemistry of this initial prokaryote system which started environmental change. The difference between our approach and many other descriptions of the development of biological systems is that we tackle the system problems, organisms plus environment, only from element composition, treating elements as

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components, not from particular organic chemicals, and we assess local concentrations of many components where and when they are or are not restricted by equilibria. While doing so we shall refer to the several component metal ions in the composition of a compartment under the title of the ‘‘metallome’’ while the small non-metal components will be included under the heading ‘‘metabolome’’, both being necessary in addition to the ‘‘genome’’ and the ‘‘proteome’’ to define a cellular composition. The Nature of the Cytoplasm The essence of life in chemical terms must be describable by those features which are common to all cells. We then set aside treatments of complex organisms and evolution while searching for basic necessities, turning to evolution of greater complexity later. In essence this requires a description of the common features of the cytoplasm of all cells in a contained system of chemical flow at ‘‘so-called’’ rest. Rest is an active, i.e. dynamic, homeostatic condition. Note that we are avoiding the problems of development, reproduction, dormancy and death while we ask what is common to all cells while they remain in such a steady ‘‘rest’’ state of life which can include growth at fixed composition. Some features of the cytoplasm of all cells are obvious such as the presence of several classes of polymers including DNA, RNA, proteins, lipids and saccharides all derived by reduction of basic C/H/N/O compounds. There are few oxidized non-metal centres present. We have no grounds for saying that there are other forms of life not based on these large molecules, but we note immediately that the synthesis of each polymer is restricted. Other essential features are uptake and loss of a variety of small organic chemicals also containing the elements C/H/N/O/P/S, pathways of synthesis and degradation, the intake of energy in order to make the above polymers, and integrated control of the whole. The overall controlled functioning has common chemical features of energy utilization through thioesters or bound phosphate in such units as ATP, a derivative of pyrophosphate. These cytoplasmic activities, which differ only in particulars from organism to organism, all

maintain the above major common polymers and they are all based upon the controlled handling of synthesis using these six chemical elements. Now these are not the only essential element components in cells and a further starting point for the examination of the common ground of all cells (in fact for that of any chemical system) is full quantitative element analysis, of the cytoplasm, looking for those analytical features, basic component concentrations, which are general to all cells, and treating their free ionic or bound simplest molecular forms, e.g. sodium as Na+ and carbon as CO2. Finally all cells have similar internal cytoplasmic messenger networks as well as reaction paths and these too are based on certain organic molecules and free ions. Hence through element analysis we have a first impression of survival strength common to all cells against the basic units of composition. For a detailed account of this analysis and full references see our books (Frau´sto da Silva &Williams, 2001; Williams & Frau´sto da Silva, 1996 and 1999). Element Analysis in the Cytoplasm As stated earlier, top-down enquiry into the nature of any chemical system must start from element analysis followed by the examination of compounds, their concentration, and their location in space, and then any equilibria between them. The simplest form of any element present can be treated as one component no matter how many additional non-equilibrated compounds have to be included. The structure and the turnover of these products from the elements in a steady state or in an equilibrium are not essential to this enquiry. This is true for nonliving as well as living systems. Fig. 2 gives some common features of the element components of the cytoplasm of probably all cells in terms of free element concentrations. We find in fact that in all organisms we need to describe the concentrations in the cytoplasm of about 20 free elements obtained from the environment in order to describe this central part of a living system. Certain elements are invariably reduced (Na, Cl, Ca) while others (K, P) are increased in concentration in the cytoplasm relative to values in the sea (see below). Perhaps surprisingly, at

EVOLUTION WAS CHEMICALLY CONSTRAINED

Fig. 2. The approximate distribution of free metal ions in the cytoplasm of all cells. This distribution is a necessity for the maintenance of the common metabolic paths of all cells.

first sight there is already the underlying suggestion from the constancy of the concentrations of the elements (components) that once organisms evolved, based upon one central cytoplasmic compartmental system, it had to remain virtually fixed in its cytoplasmic metallome and metabolome. Why? This is not because of a coded constraint since, as we shall see, other parts of organisms have evolved with a great variety of compositions and the code, DNA, has been enormously increased in size in evolution. The code must in some way have been introduced to reflect the internal necessity for maintaining a particular basic chemical composition in the cytoplasm of all organisms apart from acting in order to provide for a mode of reproduction. (Note that a code can only represent an initial pre-existing necessity.) We take this composition as the fundamental necessity of survival. There is a second fundamental feature of the cytoplasm of all cells F the elements are present or combined very largely in reduced states, the same as or more reduced than those in the environment, at a fixed pH. As we have mentioned already the overall composition of saccharides (4CHOH polymers), lipids largely (4CH2 polymers), proteins [not far from the composition (4CHOH)n plus (NH)n], nucleotides (saccharides plus bases close to oxidation

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state zero for carbon and minus one for nitrogen) and the small non-metal molecules of the metabolome are reduced states with respect to CO (CO2) and N2 of the atmosphere. (Remnants of life present on Earth today such as gas, oil and coal are all extremely reduced as compared with atmospheric CO2 or even CO). Sulphur is found largely as RSH or S2, iron is present as free Fe2+ and other metal ions occur also as M2+ or M+ ions (Wachtersha¨user, 1988; de Duve, 1991). The cytoplasm of all cells is buffered to around pH ¼ 7, a value close to that of the early sea, and its compounds of metals and non-metals largely have a redox potential range from about –0.4 to 0.0 V dependent on this pH. The overall chemistry of the cytoplasm and in fact the general metabolism of primitive cells therefore involves energized reduction of many elements from their states in the environment. We shall turn to this energization later but we need to ask why must the synthesized organic and inorganic materials be so reduced? We believe this is the only way in which organic polymeric molecules can become of high kinetic stability in water. We may then see reduction as the primary feature of organic energized cellular chemistry where energy applied forced an increase in disproportionation and separation of oxidation states such that while parts of the non-metal elements became more reduced and stabilized in polymers in cells, a second compartment, the environment, contained more oxidized states of the elements. We shall return to which particular metal elements are essential in the cytoplasm after we have described the environment from which they are extracted and reduced. Given this background to the basic chemical composition of the most primitive cells, we can turn to the easier problem of the variables of systems that surrounded the earliest cells F the primitive atmosphere and the sea between which we shall assume elements were in mass transfer and often chemical equilibrium. The understanding of the limitations of their element contents has to start from chemical abundances on the Earth and then go to any speciation in soluble (in the sea) or in volatile (in the atmosphere) forms, that is to the availability to cells. It is availability which is the critical

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consideration in determining the survival of the life/environment system. Abundance and Occurrence of the Elements in the Earth The abundance of the elements in the universe, and the way our planetary system formed, controlled abundance on the primitive Earth, which is a metal-loaded planet (Prantzos et al., 1994). The fact that metal elements and hydrogen were somewhat in excess of non-metals such as oxygen made the initial total condensed matter reduced. (Even today Earth has a vast reserve of highly reduced metal material in its core). The intermediate and outer condensed layers were and are composed of somewhat less reduced metal compounds such as sulphides or lower valent oxides. The atmosphere containing non-metals was and is the most oxidized. The aqueous zone is neutral in this respect since hydrogen here is oxidized and oxygen reduced in H2O (Anderson & Crerar, 1993). Of the important and relatively abundant non-metal elements, oxygen appeared in solids mainly in low-valent metal oxides or mixed oxides such as silicates and in liquid water, while in the atmosphere nitrogen formed as N2 or NH3; carbon as CO (CO2) and CH4; chlorine as HCl initially (while out of contact with metal oxides), and sulphur as metal sulphides and H2S. Note particularly that much of the carbon was always oxidized to CO2. In the prevailing conditions of abundances equilibration and degree of reduction several of the non-metal elements could not be forced into reduced oxidation states such as hydrides and remained mainly in higher oxidized forms in solids or ions. Amongst them the obvious examples are phosphorus as phosphate, silicon as silica or silicates and boron as borates. (In organisms too, phosphorus, boron and silicon are always present in these their highest oxidized states.) Much of the carbon could not be reduced and remained as CO or CO2. Oxidation of sulphur and nitrogen was prevented by the overall lowered availability of oxygen due to its high affinity for metals, hydrogen and carbon. The metals which could not be reduced and were kept in their highest oxidation states occurred

in large part as simple ions in oxide lattices or soluble salts. This is the case for Al3+, Ca2+, Mg2+, Na+ and K+. Some of these readily gave insoluble mixed oxide based salts such as silicates and carbonates. The most soluble oxides then reacted with the limited amounts (by abundance) of HCl to give soluble chlorides in the sea once water condensed, e.g. NaCl and KCl. Note that those metals with a relatively low affinity for oxygen could not form oxides (oxygen was not in sufficient abundance) and they formed sulphides (e.g. of many transition metals) or metals and alloys (e.g. metals such as gold). Initially, there would have been no excess of oxygen or sulphur as elements though both were present as hydrides, H2O and H2S. All the associations in the exposed surface zones are firmly based on the restrictions of variables due to total composition (abundances) and certain equilibria at room temperature and pressure. These availabilities of the elements in particular oxidation states were extremely strong constraints upon the form an energized system, such as primitive cells could take later. Notice also that the sea had a considerable concentration of ions. Abundance has curious additional and inevitable kinetic features. Thus some light elements are very rare, e.g. Li, Be and B, due to the nuclear pathway of formation of the elements of greater mass, while elements heavier than zinc are also of decreasing abundance. Other elements of comparatively lower abundance are those with odd atomic numbers such as Cl, K, Sc and As, while there are a few atomic numbers which have special nuclear stability and therefore abundance, e.g. Fe. Thus abundances are a strong kinetic constraint on the variables of composition of chemicals in the environment and thence on cellular organic reaction systems which need many elements for reasons given below. We therefore stress the nature of the early atmosphere and sea, based on abundances and occurrence in compounds due to permitted reactions. It is this combination which generated the concentrations of the all important elements, their availability open to energisation and then formation of organic systems, which predated life or grew simultaneously into coded life. [Note that while the environment is locally

EVOLUTION WAS CHEMICALLY CONSTRAINED

heterogeneous it will be treated here as homogeneous over periods of 500 million years]. The Availability of Elements in the Early Atmosphere We summarize first the early atmosphere as outlined above. It was somewhat reducing in that it contained H2, CH4, CO and H2S in addition to CO2 (Kasting, 1993; Cox, 1995). It is difficult to say if the CO and CO2 as somewhat oxidizing gases were or were not in thermodynamic equilibrium with the reducing gases H2 and CH4 and with H2O given the uncertainty in abundances. The nitrogen content is uncertain since there could have been such gases as N2, NH3 and HCN. The exact partial pressure of these gases is unknown but it is thought that the major gases were N2 and CO2 with considerable amounts of CO. The gases themselves will have been in approximate thermodynamic equilibrium with their solutions in the sea. The Availability of Elements in the Early Sea Availability in the early sea is more difficult to discuss since it depended on the solubility of compounds, particularly salts. The vast majority of abundant metal ions form rather insoluble salts with abundant anions such as silicate (Mg2+, Ca2+, Al3+) or sulphide (transition metal ions). This leaves a few abundant metal ions, Na+ and K+, and one anion, Cl, which do not easily give precipitates with abundant anions or cations respectively. As a consequence, especially Na+ and K+, and Cl were left in solution in the sea and were limited there by the lower abundance of chlorine relative to the combined abundances of Na and K. Almost all chlorine on Earth is still in the sea or it evaporates today as NaCl. There was also present a small quantity of bicarbonate/carbonate accompanied by the metal elements, Mg2+ and Ca2+, which form somewhat soluble salts. Note that there was then no sulphate or nitrate since they were too oxidizing. Of the transition metal elements both the silicates and sulphides are all insoluble but with increasing atomic number in the transition metal series from Mn to Cu the sulphides become the dominant species and they are of particularly small solubility in

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water. The profile of solubility products in the primitive reducing sulphide system (H2S was present at approximately saturation levels of millimolar) is shown in Fig. 3. Note these insolubilities caused the virtual absence of copper and initially of molybdenum and the very low content of zinc, precipitated as sulphides, while iron as Fe2+ and Mn2+ and even tungsten (W) remained in somewhat soluble forms in the primitive sea. Figure 4 gives the approximate probable concentrations of all of the major ions in the primitive sea (Cox, 1995). It is very important to observe that it is thermodynamic equilibrium restrictions on the composition variable in these somewhat reducing conditions which limited the sea’s components. The equilibrium restrictions are the solubility products, redox potentials, and the stability constants of inorganic complexes. Thus the components of the sea at equilibrium, given in Fig. 4 in terms of the elements, with the contactable surface of Earth and with the atmosphere, themselves limited by the fixed abundance and occurrence of the elements, initially provided a gross constraint on the possible development of any other system such

Fig. 3. The solubility products of sulphides (full line) and hydroxides (dashed line). The horizontal line cuts off at 104 M free metal ion at pH ¼ 7 for hydroxides and at 104 M free metal ion at pH ¼ 7 and [H2S] ¼ 103M for sulphides.

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Fig. 4. The composition of the sea today (full line) compared with the probable composition of the primitive sea some 4.5  109 years ago (broken line).

as cellular life, see Fig. 2. The whole is an open system linking the environment and cells. In fact the early sea and early cells (cytoplasm) had many gross features in common. However the composition (components) of the sea and the atmosphere were open to change since the chemicals were not in chemical equilibria with all those that could be injected from the core nor were they in temperature equilibrium with the hot core and very hightemperature sun. The environment was open to thermal and photochemical kinetic change even before life began. The ejection from the core would be expected to produce some extra excess of reducing components now and then. Consider however the more important effect for evolution of photolysis of water possibly catalysed by iron salts in the sea: 2H2 OðcÞ-2H2 ðgÞ þ O2 ðgÞ Now the gases H2 and O2 are more reactive with other chemicals than with themselves so that the reverse reaction did not happen before hydrogen reduced some chemicals and oxygen oxidized others. This is disproportionation and separation of oxidizing and reducing components. The kinetic and sustained traps for hydrogen were mainly light non-metals such as carbon and nitrogen while the traps for oxygen were heavier non-metals such as sulphur and then various

Fig. 5. The oxidation/reduction potentials of element couples indicating the states of elements in the primitive conditions 4  109 years ago (not much above the hydrogen couple potential) and today (not far from the oxygen couple potential). Note the change in the conditions of most elements in the environment indicated by the arrows to the limit of H2 (H2O) goes to O2 (H2O). Metals are to the right, non-metals to the left.

metal sulphides such as V, Cr, Mn, Fe, Co, Ni, Cu, Zn and Mo slowly producing more soluble oxides. A feature of the states of these metals is that over a reasonable period of time they equilibrate with oxygen and amongst themselves but not with C/H compounds so that we can believe that the sea and atmosphere changed continuously through an increase in redox potential equilibrated with the slowly increasing O2 partial pressure (Fig. 5). These equilibria are strong constraints on the variables of the possible components of the environment at any one time. At the same time more reducing equivalents were trapped (stored) in organic molecules and the carbon and nitrogen compounds in the atmosphere were also oxidised so that the atmosphere became increasingly just CO2 and N2. Later still surface sulphides were oxidized to sulphate and N2 to some nitrate in the sea. Figures 4 and 5 together indicate the inevitable progression of oxidation states in the environment, that is the changing of the available environmental chemicals open to the metallome and metabolome of organisms from one dominated by moderate reducing conditions to one dominated by oxidation. The kinetic trapping of reduced products was to some degree in simple C/H compounds seen in

EVOLUTION WAS CHEMICALLY CONSTRAINED

such materials as oils. There were then inevitable new phase separations (compartments) in the liquid temperature range. Turbulence of the mixture of oil and water could have led to the formation of small isolated compartments (vesicles) and they could have become separately energized since they would absorb energy unequally especially if some oils were unsaturated before life started but in this article we do not need to discuss this possibility (Segre´ et al., 1999). It is however extremely important to realize that the only way in which organisms could arise was in this reduced compartment and was from element combinations even more reduced than the environment, see the cytoplasmic content above, so that life itself enforced the increasing disproportionation and separation of oxidized (environment) and reduced (organism) chemicals demanded by the sun’s energization of the Earth, Fig. 6, and see Note at the end of this article.

Fig. 6. The general chemical relationship between the crust and biosphere of Earth in part driven by the energy of the sun and the core of Earth.

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The Chemical Path Evolution of Life had to Follow We have now described the basic unavoidable reactions of the primitive cytoplasm and of the environment and our next task is to link the two in cells. We shall presume that the redox flux in cells using energy and probably H2S at first, created the internal reduced sulphur (thiolate) and Fe2+ ions and bound hydrogen (NAD) chemistry which yielded polymers through reaction with CO and N2, and that the system rejected elementary sulphur to the environment. It may be also that tungsten was used not molybdenum. The reactions in cells led to a set of sugars, lipids, proteins and nucleotides. Thereafter this system could not change greatly and certainly did not do so. The cell system was in a homeostatic condition through exchange of material, energy and functional instructions between chemical pathways. Messages were based upon mobile units such as Fe2+, Mg2+, substrates and coenzymes (see Williams & Frau´sto da Silva, 1999). The system somehow became coded via RNA and DNA, the syntheses of which are dependent on the reactions in the cytoplasm. Coded reproduction is conservative and stabilizes organisms and this became essential for the continuity of the cytoplasm throughout all time, but we are not concerned here with coding. However, the nature of the environmental reducing agent used by organisms could be and was later changed from H2S to H2O taking advantage of the vast availability of hydrogen in water. The dramatic result was that reduction/oxidation changed from that between the essential NAD+/NADH redox potential and that of the oxidized product Sn (environmental precipitate) from H2S to that between NAD+/ NADH and the oxidized product O2 (environmental gas) from H2O, that is from NAD+/ NADH 0.35 V S2/H2S 0.0 V, to NAD+/ NADH 0.35 V O2/H2O +0.8 V. This change could have been a disaster for the reductive organic synthesis in organisms if O2 or new oxidizing agents, no matter how they were derived from O2, had immediately entered and been activated in the cytoplasm. What happened first was essentially cytoplasmic protection from dioxygen and its immediate reduction products

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such as H2O2, that is further rejection of oxidizing units from within early prokaryotes to the environment. Once protection was secure, then advantageous use of H2O2 and O2 and the other products which were generated by the general equilibration of redox equivalents in the  sea, such as SO2 4 and NO3 , could follow but only outside the cytoplasm, in new compartments. (These products were also a large possible new energy source due to these oxidized materials, Table 2.) We shall state that it was just this requirement to come to terms with oxidized materials which forced primitive cells to increase the number of compartments in order to survive in the new environment. This is the switch from prokaryotes to eukaryotes. Moreover, as the redox potential of the environment increased and equilibrated with the dioxygen partial pressure, so a succession of further oxidized inorganic products evolved, Fig. 5.

Each and every new oxidized chemical was firstly a danger to organisms and then of potential value as a source of new chemistry as well as of energy due to the redox gradient relative to the cytoplasm, Table 3. At the same time many essential elements which were necessary in a reduced state in the cytoplasm had become oxidized in the environment, and they needed to be scavenged and then reduced, e.g. iron. Thus the changes in the environment were a burden at first leading to a gain later. Finally, as new chemicals were made in new compartments, they had to be controlled in concentration so as to be cooperative with the chemistry of the cytoplasm. Only through communication was it possible to use the new chemicals fully in new oxidative chemistry in new compartments, since any such chemistry had to become coded and reproducible using instructions from the reductive chemistry in the cytoplasm. This

Table 3 Involvement of elements in homeostasis during evolution Primitive anaerobic prokaryotes

Early (anaerobic) single-cell eukaryotes (additional features)

H, C, N, O, P, Se (hydride transfer) substrates and polymers

Later (single-cell) and multi-cellular eukaryotes (aerobic) (additional features) Oxidized chemical forms Se peroxidase

H+, Na+, Mg2+, Cl, K+, Ca2+ exchangers and ATP-ases

Inner vesicles acidified and with more Na+, Ca2+ and Cl

Outer filaments and signalling between cells, Na+/K+-ATP-ase Organic hormones Iodine hormones

Ca2+ structural

Ca2+ vesicles and inner filaments and signalling via calmodulin

Annexin, S-100 and other Ca-binding proteins

H+, P, S, Fe signals

IP3 signals

S- and P-based hormones

W enzymes

Some Mo enzymes

Only Mo enzymes

Mn, Fe, Mo, Se low redox potential enzymes

(O2 release from Mn enzyme photo-system)

High redox potential enzymes

Ni enzymes (H2, CO)

Plants only

Ni (urease)

Animals only

Co (B12) (Zn enzymes)? Zn structural No Cu

Zn enzymes increase

Not in plants Zn enzymes in vesicles Zn signalling (DNA) Cu enzymes

EVOLUTION WAS CHEMICALLY CONSTRAINED

communication again needed new chemistry and clearly this could be based only on oxidized or previously rejected units outside the cytoplasm in temporary communication with the inside. The chemicals inside the cell were already used for selected purposes and could not perform this function. This overall direction of evolution was inevitable and led progressively to eukaryotes, and then multi-cellular organisms. We insist that it was the need for reduction in the cytoplasm which drove oxidation of the environment which in turn fed back to organisms forcing evolution of complexity. Much though Dawkins’ image of the watchmaker who is blind may be useful in the description of species evolution, (see below), we see that the overall activity of the watchmaker was constrained by the nature of changing chemicals and the thermodynamic equilibrium conditions of the environment. Together with the unavoidable interior reductive chemical workings allowed in the primitive watch, some of it also constrained by equilibria, they enforced a one-way progression in extra compartments. Hence, as a maker of a code (and species) the watchmaker’s possibilities were perhaps random (there are other possibilities; Caporale, 1999), so that blind he or she may have been in this respect, but in the general direction the work was guided. Life was in a physical chemical tunnel and there was only one way to go. To summarize: the tunnel of opportunity was constrained by the need to incorporate unavoidable new chemistry within the organization which could only be achieved using: (a) more oxidative chemistry extending chemical variety (not in the cytoplasm) from that limited by H2S to that limited by H2O oxidation; (b) more compartments to extend the use of space and avoiding conflicting chemistry. We shall see the need for this development more explicitly in the next section; (c) communication between compartments so as to maintain organised control by DNA; (d) energy capture from novel sources, principally from O2; (e) more extensive feedback controls linked to an increase in DNA and to the environment. This linkage also increased protection.

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Complexity of higher and higher order arose inevitably though its exact timing is unknown. We turn next to the chemical element details of this inevitable progress from primitive to higher organisms leading up to human beings, when a quite novel purposeful chemistry developed. Initial Developments of Prokaryotes Above we have described the essential features of the basic chemistry of the cytoplasm of all cells. Life required other unavoidable chemical element features forced upon it by the environment apart from reductive organic syntheses even in the most primitive cells known to us. These are: (1) rejection of Na+ and Cl in order to control osmotic pressure relative to that of the sea and uptake of K+ in order to balance anion charges in the cytoplasm (note that most biomolecules are anions at pH ¼ 7); (2) rejection of Ca2+, to avoid precipitation of organic anions in the cytoplasm; (3) energy supply dependent on environmental chemical sources or light, and using iron in electron transfer centres, electron transfer, forced by energy, caused disproportionation of reduced and oxidized chemicals; (4) a supply of bulk non-metal elements C(CO2, CO, CH4), N(NH3, HCN, N2), O(CO, CO2), S(H2S) and possibly H(H2, CH4, NH3) most of which were incorporated in reduced states together with PðHPO2 4 Þ: Their sources are in brackets; (5) a complement of trace metal catalytic elements all in reduced oxidation states which we list as Mg2+, Mn2+, (Ni2+), (Co2+), (Zn2+) and Mo or W, and the trace non-metal Se (those in brackets may not have been essential initially). They were required for weak (Mg2+) and strong (Zn2+) acid catalysis and for structural purposes, and for transfer of H, CH 3 (Ni, Co) and O (Mo or W). Details of the use of each metal and non-metal are given in books on biological inorganic chemistry (Frau´sto da Silva & Williams, 2001).

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It should be understood that we are not denying that in the most primitive cells we know both the organic and the inorganic chemistry in the cytoplasm of the cell were controlled by a code and feedback of several kinds, as well as by thermodynamic and kinetic constraints (Eigen, 1996). The DNA provided the coded instructions for the synthesis of all the proteins of the cell but it alone could not give their concentrations. The concentrations of the proteins were decided by feedback from the products of reactions and from the reactants themselves to the code. The reactants were in pathways of reaction, catalysed by proteins and metal ions, as were the syntheses of proteins and the generation of required energy. Both were controlled by feedback and feedforward from substrates to their own and other pathways and to and from the pathways of energy supply, e.g. from ATP. Now the substrates were dependent ultimately on supplies of the above basic chemicals from the environment and this uptake was largely managed by pumps. Pumps were controlled by feedback from internal concentrations of those basic chemicals and energy supply (ATP) which may also control catalytic activity of proteins. There was then an intense feedback/feedforward network controlling homeostasis F correlated rates of uptake and production of all essential ions and molecules and energy supply F to keep a steady state of flow (Frau´sto da Silva & Williams, 2001). Here the most important inorganic messengers in the feedback of the primitive cell were Mg2+, HPO2 4 and pyrophosphates (ATP, etc.) which controlled energy distribution, and much of metabolism; Na+, K+, Cl which controlled cell osmotic and charge stability, Mg2+ and possibly some Mn2+ and Zn2+ which manage 2 acid/base catalysis; Fe2+, WO2 4 ðMoO4 Þ; 2+ 2 and S which controlled reductive Mn metabolism. Other elements may well have had minor control roles. We are deliberately leaving on one side the more intricate and difficult controls due to substrates and products that is the organic cellular chemistry of C, H, N, and O. We shall call this cytoplasmic network the primitive messenger system but notice that the system of cell chemistry we are describing cannot be divorced from the environment any more than it can be separated from a code. All the

basic chemicals and energy come from the environment and this remains true to this day. Moreover, as stated, the cytoplasmic chemistry has changed very little of necessity. Notice also that at every stage of development of chemistry, or the use of space, organization can only be maintained by communication in a feedforward/ feedback system. While there may have been other required elements which ought to be included such as B, Si and V, the complement of essential elements is clearly close to 20 even for the most primitive cells known. This diversity of elements covering 15 groups of the Periodic Table opens the system to almost the whole of the variations of chemistry, within the limitations of acid/base and oxidation/reduction in aqueous media. During the course of the first one and a half or two billion years, the chemistry of these elements in the environment changed slowly as did the nature of the prokaryotes. Because life’s chemicals are generally more reduced than those in its environment, oxygen concentration rose to perhaps one-tenth of its present value and then equilibrated with the states of virtually all the elements in the environment in this long period. By this time the availability of the elements from Mn2+ to Zn2+ (excluding Cu) increased to considerable concentrations as sulphides changed to oxides while that of Fe2+ fell equally by many powers of ten, precipitated as Fe(OH)3. The nature of the non-metals changed to C(CO2), N(N2 and NO 3 ), O(CO2, O2 and 2 Þ; SðSO H2O), Se ðSeO2 4 4 Þ while H as H2 and CH4 disappeared, see Fig. 5. The chemical tasks of incorporating many elements became much more difficult, clearly requiring increasing complexity of organization, but the sources of energy increased. The change was slow since the reducing buffer capacity of Earth was very large. All these chemical changes were brought about by the production of O2 by organisms. This we believe was due to an inevitable chemical switch from the primitive use of H2S to that of H2O so as to provide a (larger) reducing capacity. The change to the use of H2O for reducing equivalents did require a higher energy source and a new catalyst but both were available F light from the sun and manganese

EVOLUTION WAS CHEMICALLY CONSTRAINED

from the environment. As the environment became oxidized as described above, over say one or two billion years, there were inevitable changes in the chemical pathways in prokaryotes which we give in the order in which the environment changed, Fig. 5. (1) NH3-N2, required difficult N2 incorporation using molybdenum which had become MoO2 4 in the environment, or vanadium. (2) HS -SO2 4 ; provided an energy source using the reversal of this reaction as well as new organic sulphated components but removed an easy source of sulphur. (3) Fe2+-Fe3+ locked in Fe(OH)3, required scavenging for iron. (4) Flavin and Fe2+, in several new and old complexes in cells, acted as catalysts for oxygen incorporation making possible a new range of organic molecules both unsaturated and with oxygen bound such as in cholesterol, as well as some new inorganic compounds. (5) HSe -SeO2 4 made selenium scavenging necessary but organic Se itself became used in oxidation catalysis or as 4SeO. (6) Zn2+ became more useful as an acid catalyst due to increased availability following the oxidation of its sulphide. In eukaryotes there are literally hundreds of zinc proteins (see Frau´sto da Silva & Williams, 2001). Different species of prokaryote emerged able to utilize one or more of these changes. In fact this evolution of new species with exchange of material was almost unavoidable in that it reduced complexity in single organisms. Organisms were created which could (a) use N2, for energy, the nitrogen fixers, (b) use SO2 4 sulphate bacteria, (c) use light, photosynthetic bacteria, (d) use oxygen, aerobes. All of them had still to accumulate iron, molybdenum, selenium and various other elements in difficult conditions and many came to use more zinc as a Lewis acid in structure and catalysis. We see the effect of the environment changes exerting a strain on primitive cells and a pressure to evolve. This strain could be and was lessened by the use of compartments for reactions, the periplasm in prokaryotes and multiple vesicles in eukaryotes to which we turn.

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The Evolution of Eukaryotes What could be called essential activities for life, the uptake and incorporation of the elements in useful forms became more difficult as the oxidation of the environment increased and, in prokaryotes, was dispersed into different prokaryote species with no correlation of their activity, as mentioned already. The pressure to evolve cells containing an optimal number of these activities under central control was the only new direction evolution could take. The very fact that it was dangerous to incorporate oxidized components in the cytoplasm then forced the evolution of single-cell eukaryotes, where oxidative metabolism is kept in vesicles or organelles away from the cytoplasm. The cytoplasmic chemistry is based upon reduction and oxidizing agents are a threat to DNA. Even then we find that when eukaryotes evolved, see below, they were split between (a) photosynthesizing organisms, which became plants, (b) non-photosynthesizing aerobes, which became animals, (c) nitrogen-fixing organisms, still bacterial and basically anaerobes, (d) a few anaerobic organisms largely bacterial, which were forced to inhabit zones away from oxygen. Some relief from the problems of increased complexity and slow reproduction was had by utilizing symbionts (Margulis, 1999) or by digesting prokaryotes. Photosynthetic, oxidative and nitrogen fixation activities were confined to the new or old separate compartments: mitochondria, chloroplasts, internal organelle symbionts, and external N2-fixing symbionts respectively. However, overall the different organisms in (a) became mutually dependent. There is a demand for this cooperative ecosystem to reduce complexity in any advanced single organisms. Meanwhile the involvement of elements in cells increased, Table 3. The survival strength of the eukaryote single cell demanded too that it could both scavenge for food and protect itself better than the prokaryote since it was a much larger, more

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complex cell with a correspondingly longer lifetime. These properties were based on the production of a more flexible membrane system unimpeded by a wall but yet more stable. Membrane stability was achieved by the incorporation of the oxidized organic compound, cholesterol (absent in prokaryotes), aided by adjustable new filaments. Greater protection came about through exocytosis of poisons and hydrolytic enzymes from vesicles to damage and aid digestion of prokaryotes, while endocytosis allowed engulfment of large particles. We must see that the difficulties of handling the changing environmental chemicals due to oxidation, that is the difficulties of increased organized activity in one compartment, forced developments of both multi-compartment prokaryotes and of eukaryotes. However, the eukaryotes needed and gained by one further development: a communication system, for without it there are no advantages in concentrating activities in many compartments in larger organisms. The communication mode adopted by eukaryotes depended in part on an extension of the internal messages in the cytoplasm described above and based in part on iron (Fe2+), magnesium and ATP, as found in the earliest prokaryotes in addition to the introduction of the newly available zinc. Of course there were also more uses of all the non-metal mobile messenger units internal to the cytoplasm in the larger cells, but the important novelty required was communication between new compartments in a cell and knowledge of the outside environment to increase protection of and scavenging by a longer lifetime. The connection between them was made largely by pulsed calcium ion flows into the internal compartments from outside the cell or from an internal compartment stores in separate vesicles which were used to stimulate a multitude of activities internally including basic cytoplasmic metabolism, endocytosis and exocytosis and those of the dehydrogenases of mitochondria and the photosynthesis activity of chloroplasts. The entry of this metal ion, calcium, into the cell cytoplasm followed from novel sensor responses to external events (Carafoli & Krebs, 2000). The communication between the outside environment and the internal compartments generating motility through

Fig. 7. The network of Ca2+ flows in a modern organism linking the external, the vesicular and the internal cytoplasmic solutions where H is a hormone, P a pump, F a filament, N the nucleus, M a mitochondrion, V a vacuole, ER the endoplasmic reticulum and R1 a receptor. The concentrations are external and vesicular 103M and cytoplasmic 107M and corresponding protein binding constants are 103 and 107M1 respectively. The earliest system of this kind appears in single-cell eukaryotes which have no Na/K message or externally controlled extracellular fluids but most of the other response systems.

the use of new filaments was also based overwhelmingly on the fast inward pulses of calcium followed by its immediate rejection, Fig. 7. It is not possible to see a chemical alternative to this functional value of calcium given the nature of the environment and the internal chemistry of the system it had to activate (Frau´sto da Silva & Williams, 2001). The prokaryote cytoplasm had been forced to reject Ca2+ down to [Ca2+] o106M with an external [Ca2+] of around 103M, in order to protect the organic molecules in it, with the consequence that this element became the ideal in and out messenger with its fast diffusion, adequate chemical binding strength and fast on/off rates (Williams, 1970). No other available element or compound was of comparable value in these respects. To do

339

EVOLUTION WAS CHEMICALLY CONSTRAINED

otherwise than to use Ca2+ as a messenger would be like higher animals doing without the use of sound in messages. The calcium communication network in modern eukaryotes is extremely extensive, reaching to mechanical, chemical and energy changes and developed steadily in evolution, Fig. 7. It seems clear therefore that the development of organization was driven inevitably by (1) the dispersal of chemicals into different oxidation states caused by disproportionation and separation when organisms took the reduced and the environment took the oxidized states of chemicals initially (Williams & Frau´sto da Silva, 2000), (2) the chemical gradients of inorganic elements demanded initially by the nature of the organic chemistry of the original cells (Goodsell, 1998), and (3) the advantage for survival which resulted from the use of (1) and (2) made possible by compartmentalization. The process of environmental chemical change at first was very slow due to the large reducing capacity of the original environment F hence the slow appearance and initial evolution of eukaryotes. The further and final developments could be much faster once the reducing buffer capacity of the environment had been largely overcome. Oxygen pressure rose relatively rapidly towards its present levels established some one to a half a billion years ago, Fig. 5, and new types of eukaryotes evolved dependent on further new oxidative chemistry. But note also that at first many of these oxidized chemicals were poisons, due to the very nature of cytoplasmic chemistry, including such elements as the free zinc ions in higher concentration. The harder the element was to oxidize from its source, say in sulphides, the later it was released and the more poisonous it was for example in the sequence ZnoCu; Cd oHg and Pb: Moreover, oxidized non-metal elements which were also formed are often difficult to in corporate, such as N2 ; SO2 4 and NO3 and some are poisonous, especially nitrate and halogenated compounds. The possibilities for organisms were to reject them or to find a use for them in a new compartment but usually not in the cytoplasm. In the cytoplasm there

was possible use for some non-redox elements in strongly bound forms, e.g. zinc. Oxidation and the accompanying poisonous chemicals thus drove the evolution of new chemistry and compartments of necessity and continuously. We next look at how this development, together with the unavoidable increase in complexity of the chemistry, made it more and more advantageous to evolve specialization in given organisms with mutual dependence in an ecosystem. The Evolution of Multi-cellular Organisms PLANTS

The next step for eukaryote organization after the internal vesicle or organelle (symbiosis) development was the fuller use of space external to its outer membrane. This required both new, relatively rigid, external filaments and new external messengers. We know that this progression began in plants and sponges. The cell–cell structures allowed greater access to light, occupation of the land and so on. Primarily we see this new structure in the production of lignin where hydrogen peroxide, an increasingly available chemical as O2 increased, is used by extracellular enzymes, initially required to remove this poisonous oxidant, employing a combination of external haeme enzymes and manganese ions. Later in some plants the lignin oxidation uses copper (laccases) and we turn to the extensive use of copper shortly. Notice that the lignin in cellulose is a dead product F wood F holding cells in fixed positions. The new plants and fungi needed also a chemical communication system between cells, organs, such as root and bud, leaf or flower (seed). These new chemicals, including hormones, were often organic molecules produced by oxidative chemistry, Table 4, and had to go in low concentration into the cytoplasm to reach the code. Note that of necessity they had to differ from internal messengers and calcium to avoid signalling confusion. A differential plant cell remains able to revert and the final conversion to a fully differentiated multi-cellular system awaited the coming of animals. The plant chemistry results from the use of oxidation which reached its final condition some half a billion years ago. This

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Table 4 Organic messengers produced by oxidation Messenger

Production

Reception

Destruction

NO Sterols Amidated peptides Adrenaline d-OH tryptamine Thyroxine Retinoic acid

Arginine oxidation (haeme) Cholesterol oxidation (haeme) Cu oxides Fe/Cu oxidase Fe/Cu oxidase Haeme (Fe) peroxidase Retinol (vitamin A) oxidation

G-protein Zn fingers (Ca2+ release) (Ca2+ release) (Ca2+ release) Zn finger?n Zn finger?n

Haeme oxidation? Haeme oxidation Zn peptidases Ca enzyme? Cu enzyme? Se enzyme ?

n

In the nuclear receptor superfamily of transcriptional receptors.

condition could be a final steady state possible in which use of energy in organic chemical synthesis is offset by spontaneous combustion as postulated in the Gaia hypothesis (Lovelock, 1988) but there are other possible limitations, see below. ANIMALS

The animal kingdom is quite unlike that of the plants in that while the plants respond to the environment through multi-cellular organization they are very generally not motile, pro-active. Plants cannot move around to search space neither can they forage to any great extent. Here the difference between plants and animals can be compared with the difference between prokaryotes and unicellular eukaryotes. The unicellular eukaryotes all evolved to use the prokaryote, either foraging to eat it or incorporating them in a useful symbiosis or ecosystem (Margulis, 1999). The eukaryote then required sensing apparatus to gain full advantage of its activity and to protect it. For this purpose it used the calcium messenger system as described. The foraging animals, which have no direct source of energy, equally needed sensing and foraging ability coupled to movement in space and the ability to utilize inert components outside themselves for protection F burrowing or building. Many of these reactions had to be fast in order to be protective from competitors or predators. The fastest possible responses to enable these activities, dependent upon mechanical power, were developed in the form of muscle and a nervous system with electrolytic not chemical messages. Once again this development

relies on putting to use a simple chemical demand placed upon the most primitive prokaryotes by the environment. This is the energized separation of Na+, K+, Cl, where Na+ and Cl were rejected. These are the only elements in sufficient free ion concentrations in the sea to give a strongly electrolytic conducting solution. They, with additional use of Ca2+ gradients across the cell membrane, became the fast message system of animals (Williams, 1970). Just as in the case of the eukaryote development from the prokaryote using new filaments, and of the development of plants using lignins, so new animal cellular constructs were necessary in the form of connecting filaments for muscles and elongated neurons. Clearly, the animal construct required permanent differentiation of cells and organs in a more or less rigid matrix. It is here that an element which was the last to be released from its sulphide came into play F copper, together with internal mineralization. The release of copper from its sulphide was a very late event in the oxidation of the environment since copper (cuprous) sulphide is extremely insoluble. Initially like all novel elements in the environment, such as zinc, copper would have been a poison rejected by cells but as is true of much of evolution rejection leads to the possibility of external use, see Na+, Cl, Ca2+ and Zn2+ above. Copper proteins became the external enzymes for the oxidation of organic molecules (Malmstro¨m & Leckner, 1998). Note that they are preferred over haem oxidases and peroxidases in several reactions in all animals outside cells. These copper oxidases use oxygen not hydrogen peroxide which as stated is used

EVOLUTION WAS CHEMICALLY CONSTRAINED

together with haeme enzymes and manganese outside the cytoplasm, especially in plants, and in so doing animals avoid the risk of chemistry which is undoubtedly one cause of aberrant growth, cancer. (Metastasis does not occur in plants.) The major relevant significance of the copper enzymes in animals is that they were employed to cross-link extracellular proteinaceous materials such as collagen and chitin. It is these firm living structures together with bones, Fig. 5, which are turned over (contrast lignin in plants) while they support the cylindrical nerve and muscle cells in more or less permanent organization. However, the organisms must grow with closely fixed shape and form, and therefore a ‘‘cut, grow and paste’’ mode of organization developed. Rejected zinc proteases originally designed for digestion was used for cutting. Hence the combination of zinc and copper enzyme activity was a critical feature of animal development. They were also critical for the extension of cell/ cell messenger systems. The system of organs needed further new long-term extracellular message systems to regulate its functions and to maintain homeostasis. Since all the available inorganic elements had been pressed into use, further variety could only be increased by new chemical transformations of organic molecules, now stored for fast release or just released continuously and slowly from vesicles in cells. The obvious chemicals to develop were oxidized organic molecules held in vesicles. Examples of the use of oxidized molecular message systems in higher animals are given in Table 4. Note how iron is used in the cytoplasm but copper and zinc are used in vesicles or extra-cellularly in the synthesis, metabolism and signalling by these molecules. A striking different example is the increased use of zinc fingers in transcription factors sensitive to oxidized organic messenger molecules. It is difficult to see these developments except as inevitably dependent on the changes in the environment. A brief note is in order concerning the introduction of one further group of chemicals F the halogens F since they increased protection and allowed further new messengers to be produced. The production of halo-peroxidases in

341

many eukaryote and later prokaryote species is for protective killing of invading organisms or for the production of halogenated poisons. They can only be made using quite high oxidizing conditions, Fig. 4. The use of iodine is special however in growth hormones such as thyroxines in animals, and it is interesting to note that removal requires the oxidative chemistry of selenium enzymes. Iodine and oxidized selenium compounds became available at approximately the same time in evolution, Fig. 5, after the development of eukaryotes. Thyroxines also bind to zinc fingers. In passing observe how selenium too is increasingly used in protection. Throughout the evolution of multi-cellular organisms, plants and animals, we observe two striking organizational features. First, their complexity is relieved by symbiosis. Plants do not fix nitrogen and animals depend on plants for all minerals and many organic molecules. Second, mineralization of the exterior and later the interior of unicellular and multi-cellular eukaryotes increasingly put to use the availability of calcium and the insolubility of its simple salts. The Full Use of Chemistry and Space: The Evolution of Human Beings The final step of purely biological evolution came through one further need of advanced organization in large bodies containing organs. Of the two major types of such organisms, stationary and motile, the motile organisms required a coordinated fast response system between their several sensing and mobilitygenerating organs in order to scavenge rapidly and to respond to danger. In animals there developed the brain from nerves allowing strong fast coordinated interaction with the environment with minimum feedback to genetic structures. Slowly but surely the animals became less and less dependent on synthesis coded by genes to supply the necessary control system for survival in the environment. Instead they used information stored in their brains to find and then to feed off other organisms and then to manipulate the chemistry in space external to themselves. Moreover they reduced complexity

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by further intense internal symbiosis. The most evolved animal is the human being who has the least capacity to manage the essential chemical processes. Humans are not able to synthesize a full range of amino acids, lipids and sugars apart from their need for vitamins and minerals. They however have the greatest capacity to manage the environment and other forms of life. Sophistication and dependence as earlier in evolution are essentially correlated. Humans have then used their brains to push still further the evolution of the life/environmental chemical system and its spatial organization, started four billion years ago in one inevitable direction. The final chapter of evolution will be therefore the further extension of the use of novel chemicals and of space to their limit. The application by human beings of chemical compounds produced over the whole range of the elements (100 instead of the 20 required by cells), employing novel oxidation but particularly reduction reactions and new sources of energy used externally, is moving to this limit. The uses are in new forms, in protection, message transmission, transferring goods, etc. F all the activities of mankind based upon the power of the brain, in effect a second coded store. In particular note the use of metals, highly reduced forms of elements, for constructs and then in message transmission and reception. Many useful plastics are also highly reduced carbon compounds. The developments are close to utilizing all available space. Genetic modifications could not have generated these developments. Nor could they have found new sources of energy such as that from atomic piles. Finally genes could not have used space in the way humans do. Even so the developments are in the same logical driven directions as those of biological genetic evolution F novel chemistry in extended use of space with a communication network. Unfortunately, human organization has not yet found a good control over cooperative activity and competition in society, much though the exploration of chemistry and space are approaching completion. This may put the global ecosystem at risk so that the final state may be not the optimal unless judicial action is taken.

Overall Inevitability of Chemical Evolution in Ecosystems The basic drive of all evolution is the transfer of energy from a hot body, the sun, to a cold body, the Earth, Fig. 6 (See Note at the end of this article). In this process the temperature of Earth is raised and the free energy content of chemicals is increased. This chemical storage of energy lies inevitably in the separation of the stable oxidation states of many elements as a result of the storage of reduced compounds in kinetically stable, mainly non-metal systems (especially organic carbon chemicals) in organisms and of the consequential storage of oxidized chemicals in the environment. The ever-evolving interaction between the two as they progressed can be best managed in a cooperative system of many kinetically isolated compartments, cells and organisms leading to an ecosystem of organisms and the environment. We are led to believe that the drive to the most effective set of cooperating, not competing, independent organisms is such that together with the environment it generates an optimal total uptake of energy (see Corning, 2002). Each advance of complexity provided the opportunity for greater species diversity. The whole evolved slowly until the appearance of humans. Humankind has pushed rapidly the variety of chemical and spatial organization further along the same path, and by new means of chemical synthesis in space external to itself. However, humans must observe that the optimal possible retention of free energy, the drive behind evolution (see Note at the end of the article), is based on a fragile steady state open to disturbance by the introduction of new chemical elements for better or for worse. It is worth noting how different this system’s approach is from the conventional molecular thinking, Table 5, which leads to no logical thinking concerning the future or the past. However, to proceed further we still need knowledge of how organisms became codified and how the code responds to the environmental change. Note In a system where energy flows from a constant high-energy source to a constant

EVOLUTION WAS CHEMICALLY CONSTRAINED

Table 5 Approaches to chemical interactions (variables) Molecular approach (stationary)

Systems approachn (dynamics)

Composition Sequence Structure (shape) Dynamics (internal) e.g. DNA, proteins, polysaccharides, lipids and small molecules or ions

Composition Concentration (C) Temperature (T) Pressure (P) Spatial divisions (phases), compartments Time (t) Proteomics Genomics, interactive dynamics Metallomics Metabolomics

n

Stationary systems may show emergent properties on change of variables, such as melting points and allotropy, and can be defined by combined thermodynamics functions such as DG, DH, DS as well as by T, P, and composition at equilibrium. Time-dependent (irreversible) systems are not easily described by functional terms.

low-energy sink then any inserted intermediate body will retain a limiting portion of the energy to reach eventually a steady state of optimal energy retention. The Sun, the cold cosmos and Earth are in such a positional relationship. As a consequence, the atmosphere of Earth has reached and held a fixed temperature gradient in a steady state for billions of years. This retention is one of optimal free energy of a physical system. Clearly, chemicals exposed on Earth will also be driven towards such a steady state of optimal energy retention and maybe it was progress towards this state which forced life to start and then for evolution to proceed as it has done in a general inevitable way. The whole system may have come close to its final steady state some 5  108 years ago (see Corning, 2002; Jorgensen et al., 2000). However today we may be on the way to replace it by another due to the impact of human activity. We are extremely grateful to one referee who went to great lengths to improve this paper. We regret that we have no way to make a personal acknowledgement.

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