Water: life's matrix

Philip Ball
Nature
4 Crinan St
London N1 9XW
UK
[email protected]
This is the (somewhat unpolished) text of a talk delivered
for the University of Utrecht, 2002
It is accompanied by a Powerpoint presentation
________________________________________________________________________
Water is the archetypal liquid: in the Aristotelian scheme of elements it represents all
things that flow. Yet we now know that water is profoundly unusual, and defies many of
the expectations of liquid-state theory. These peculiarities may make liquid water
uniquely suited to act as the matrix of life. At the same time, they make it hard to discern
exactly how water acts in this capacity. The nature of water in the cell, and in particular
the question of whether or not it resembles bulk water, has been hotly debated by
biophysicists for decades, still with no consensus in sight. Nonetheless, it has become
increasingly clear in recent years that water should be regarded as a biomolecule: not the
passive backdrop on which lifes chemistry is enacted, but an active participant. I shall
look at the question of what we do and do not know about cell water and its interactions
with other biomolecules. I shall also consider whether waters uniqueness justifies the
decision by NASA to follow the water in its search for extraterrestrial life, and shall
briefly review the status of attempts to locate liquid water in the past or present
environments of other planets and their satellites.
________________________________________________________________________
Some time ago I was asked to contribute an article to a special issue of the journal
Cellular and Molecular Biology [July 2001, 47(5)], devoted to the topic of water in the
cell. I decided that might be an interesting thing to do, but the guest editor of the volume
seemed concerned that I should be aware of what I was letting myself in for. She implied
that any statement about this topic entailed an element of risk and was likely to offend
one party or another.
This was not entirely news to me. Some of the most empassioned exchanges that Id
encountered during my years as an editor at Nature have involved the structure and
behaviour of water. Indeed, I had some kind of baptism of fire when I joined the journal
in 1988, just weeks after it had published the now infamous paper by a group of French
biologists on the memory of water. As I was coming from a research group that
specialized in the physics of liquids, that was a matter of not inconsiderable
embarrassment. A year later water was back, in the form of cold fusion. I shall not say

any more about either of these notorious episodes here; there is plenty in my book. And
even now, the polywater scandal of the late 1960s is spoken about in hushed tones and
with embarrassed glances.
These disputes about what water is and what it does are by no means limited to academic
science. I have delved a little into the extraordinary story of the Austrian forest-warden
Viktor Schauberger and his ideas about living water, expanded later by Theodor
Schwenk. This is an area where New Age mysticism blends into technological reality:
Ive heard other scientists swear that magnets really can descale kettles, and that specially
treated metal rods can alter the taste and viscosity of water with a little gentle stirring.
Recently a non-scientist friend told me about the work of Masaru Emoto, who changes
the shape of snowflakes with prayer:
The photo at the top is of a frozen water sample from the lake at Fujiwara Dam, in Japan.
The sample is described as dark and amorphous, with no crystalline formations.
But then the Reverend Kato Hoki, chief priest of the Jyuhouin Temple, prayed for one
hour prayer beside the dam, new samples were withdrawn, and the crystals at the bottom
were grown from this water.
Apparently music makes its influence felt too: when water was crystallized after being
exposed to Elvis Presleys Heartbreak Hotel, the crystals are reported to have broken
spontaneously into two.
Now, why am I telling you this? Certainly not to imply that this could be a good research
field for your new institute. No, its to remind you that studying water is not like studying
any other substance. It is not a neutral subject. All of us bring to it a wealth of cultural
associations that are probably impossible to put aside. Water is an elementsnot, of
course, in the chemical sense, but in the mythical and poetic sense, and that is not
irrelevant. Id recommend to you the book by the French philosopher Gaston Bachelard
called Water and Dreams, which explores the various poetic incarnations of water. I
believe that some such ideasof water as purifier, water as saviourlay unconsciously
behind some of the pathological episodes of water science Ive just mentioned.
Most prominent of all is the ancient and profound association of water with life. Albert
Szent-Gyorgi, the Nobel laureate biologist, famously summed it up this way: Water is
the matter and matrix of life.
In one sense, this could be taken to mean simply that water is essential for lifea notion
that is not proven but which I think is very hard to imagine as anything but true, and
whichas Ill discuss laterconditions all of NASAs current thinking for its program
in astrobiology.
In another sense, it means something more than that. Theodor Schwenks idea of living
water seems to be essentially a poetic notion unconnected to any scientific criteria,
although his supporters would no doubt disagree. But there is a very real manifestation of
living water in the form of the cytoplasmwater in the cell. Many biologists are

increasingly starting to think that there is something special about this water that sets it
apart from ordinary, bulk water.
First, some basic things about bulk water. I doubt that to this audience I have to say very
much at all about that, but it might nevertheless be just as well to establish a few points
about water as a liquid. As I say, many years ago I worked for a short time on liquid-state
physics, but I studied nothing at all about water. Why? Because a lot of conventional
liquid-state theory breaks down when it comes to water. We were warned away from it;
water was too difficult, so we stayed with liquid argon.
The reason why water is too difficult, of course, is its hydrogen bonds. From the point of
view of liquid-state theory, hydrogen bonds complicate matters because they mean that
the short-ranged, local ordering around any one molecule is dominated not by hardsphere repulsive interactions, as it is in simple liquids, but by orientation-specific
attractive interactions. These link water molecules into the well-known random
hydrogen-bonded network, for which Gene Stanley and others showed in the 1980s that a
description in terms of percolation theory could be appropriate.
What is more, the hydrogen bonding sets up a tension between the usual tendency in
liquids of close molecular packing and the requirement for molecules to be kept at arms
length, as it were, in order to form hydrogen bonds. This is the central feature of waters
structure and behaviour, so it was essential that I showed it in my book. But[Show
slide & tell how I was discouraged from drawing real ball & stick molecules in my book.]
This arrangement gives water an unusually high degree of local structure, and also a
propensity to leave lots of empty space between moleculeswhich, of course, results in
the density drop on freezing. It is also possible to ascribe to this structure anomalies such
as the 4 C density maximum and the possible existence of two different phases of deeply
supercooled water with a critical point estimated at 220 K and 100 MPa. [Phase diagram]
For cell biologists interested in water, the key question is how this unusual degree of
structuring is modified by the presence of biological macromolecules, membranes,
dissolved ions and so forth. This is the issue on which there is no agreement.
Key questions:
First of all, lets have a look at the aqueous environment of the cell. Cell water represents
70-80 percent of the cells mass and volume, and one can calculate just on the basis of
averages that the macromolecules are separated from one another by typically no more
than 3-4 layers of watera distance of around 1 nm. In this sense, there is probably little
if any bulk-like water in the cell. Just about all the cell water feels the effect of the
biomolecular constituents. The question is: what is the nature of that influence? In 1969,
Rudolph Peters had this to say:
I believe the cell to be organized not only in respect of its grosser parts such as the
nucleus, but also in regard of the actual chemical molecules of which it consists. Owing

to the microheterogeneous nature of the system, surface effects take precedence over
ordinary statistical, mass-action relationships and become in the ultimate limit
responsible for the directing of the whole, and therefore, the direction of activities.
[Proc. Roy Soc. B, 173, 11-19 (1969)]
The cytoplasm is widely regarded as a gel which seems able to maintain its integrity even
if large ruptures are made in the cell membrane. On the other hand, many molecular
biologists continue to use the paradigm of free diffusion to describe molecular motions in
the cytoplasm, and this picture often seems to be remarkably satisfactory.
A typical example of a biologists view of cell water looks something like this:
[Slide]
Structured water is a term common to the cell and molecular biology literature, but
what, precisely, is meant by it?
The idea that cell water somehow has a different structure from bulk water goes back a
long way. Szent-Gyorgyi talked about the structuring of cell water in the 1950s, and G.
N. Ling proposed in the 1960s that cell water forms organized, layered structures on the
surface of proteins. This sort of notion owes a great deal to the idea of Henry Frank and
Marjorie Evans in 1945 that hydrophobic solutes increase the ordering of water.
Proteins contain both hydrophobic and hydrophilic regions, and one needs to ask how
water responds to both. There is still no consensus in either case. Once Bernal, Pauling
and others had clarified the basic tetrahedral local ordering of liquid water in the 1930s,
discussions of water structure began to be couched in terms of an essentially crystalline
paradigm, rather than the gas-like starting point of conventional liquid-state theory. Frank
and Evans supposed that hydrophobic solutes increase this ordering in order to minimize
the disruption to the hydrogen-bonded network. They said When a rare gas atom or
nonpolar molecule dissolves in water the water, so to speak, builds a microscopic
iceberg around it.
In 1959 Walter Kauzmann realised how this sort of picture could lead to the observed
attraction between hydrophobic species in water. As two hydrophobes come together and
their ordered hydration spheres overlap, some structured water is freed and there is a
gain in entropy. According to this explanation, the hydrophobic force has an entropic
origin.
This force is of crucial importance to cell biology. It accounts for the aggregation of
proteins and is one of the main driving forces of protein foldingthe idea being that the
protein chain hides its hydrophobic regions by bundling them up together in the
proteins interior.
Kauzmanns model is still popular, but there seems to be no good evidence that it is right.
It is hard to measure water structure around hydrophobic solutes, because of their very
insolubility. But recent neutron scattering and EXAFS measurements for solutes like
methane and krypton show no clear sign of increased water ordering in these hydration

shells. There is some indication that water molecules tend to align their O-H bonds
tangential to hydrophobes, and that they may lose some rotational freedom as a result; but
there is no evidence for greater positional ordering in clathrate-like structures.
So the origin of the hydrophobic force is still unclear. An alternative explanation is that
the small size of the water molecule means that it is hard to make space for a
hydrophobic moleculethat is, there is a high free-energy cost, in both enthalpic and
entropic terms, of creating a cavity in water. This too seems insufficient as a complete
explanation. There is plenty more to be said about hydrophobic forces and the way they
depend on factors like temperature and pressure, and I will simply refer you to two good
discussions:
W. Blokzijl & J. B. F. N. Engberts, Angew. Chem. Int. Ed. 32, 1545-1579 (1993)
N. T. Southall, K. A. Dill & A. D. J. Haymet, J. Phys. Chem. B 106, 521-533 (2002)
What about hydrophilic solutes? Here the situation is no less confused. Do ions increase
water structure, or decrease it? The classic studies of Franz Hofmeister in the 1880s on
the effect of different salts on solubility of proteins has given rise to the classification of
ions as structure-making and structure-breakingor in the modern terminology,
kosmotropic and chaotropic. Small or highly charged ions, like fluoride and sulfate, are
considered structure-making. They reduce protein solubility (this is the process known as
salting-out), which is explained in a hand-waving way as being due to the greater
resistance a highly structured solvent puts up to disruption by macromolecular solutes.
Big, low-charge ions like iodide and perchlorate are structure-breaking, ostensibly
reducing water structure and promoting solubility. Chaotropic or structure-breaking ions
also promote protein denaturation and destabilize membranes.
Philippa Wiggins has constructed a theory of cell water based around the idea that
structure-makers create hydration shells of low-density, ice-like water, while structurebreakers create high-density water in which many hydrogen bonds are broken. [Slide]
Wiggins argues that these two types of water are entirely different solvents, and that they
set up osmotic and viscosity gradients in the cell which have important consequences for
cell function.
The problem is that direct probes of water structure show no support for this kind of twostate model, nor any clear signature of structure-making or structure-breaking by ions.
Felix Franks says Much has been written about this concept, much of it misleading.
Yet it does seem that waters hydrogen-bonded structure displays some sensitivity to the
influence of solutes. The hydrophobicity of D-talose, for example, is greater than that of
its isomer D-galactose, which seems to be related to the relative comfort of fitting the OH
groups into the three-dimensional jigsaw of the hydrogen-bonded network. [Slide]
All this concerns small solutes; but macromolecules can in some ways be regarded as
mesoscopic surfaces. How water responds to the presence of a surface opens up another
can of worms. Some biologists get very exercised over the high degree of structuring seen

in water next to a hydrophilic surface, as for example in these results measured by Roger
Horn and Jacob Israelachvili using the SFA:
[Slide]
But there is in fact nothing very remarkable about this kind of layering in a liquid next to
a wall. It is predicted even for simple liquids, and says nothing about waters status as a
structured liquid. The layering is due to packing effectsessentially the same thing that
gives rise to oscillatory radial distribution functions. Indeed, one can ask whether water
might be less inclined to form these two-dimensional layers since that will disrupt the
three-dimensional hydrogen-bonded network. Israelachvili and Hakan Wennerstrom
created some controversy recently when they claimed that water between hydrophilic
surfaces behaves just like a normal liquid, and that the relatively long-ranged repulsions
that have been observed between such surface, and explained by waters unusual degree
of structure, are just experimental artefects.
Hydrophobic surfaces create another controversy. Some very recent neutron-reflectivity
results by Bob Thomass group at Oxford suggest that liquid water is separated from such
surfaces by a very thin (1-nm or so) layer of gasthere is drying at the surface. This is
expected theoretically. But its been known for a long time that two hydrophobic surfaces
separated by a thin film of water seem to attract one another over very large
distances300 nm or so, corresponding to about 1000 molecular diameters. The origin
of this long-ranged hydrophobic attraction is still disputed. For a long time it too was
sometimes attributed to water structuring effects, although now there seems to be good
evidence that it is caused by the nucleation of sub-microscopic bubbles of dissolved gas,
which create menisci that pull the surfaces together. (See Carambassis et al., Phys. Rev.
Lett. 80, 5357-5360 (1998).)
A paper has just appeared in Science (25 Jan: 295, 663) by Steve Granicks group which
looks at the unusual consequences of bring together a hydrophilic and a hydrophobic
surface in water. This sounds like a rather contrived situation, but it could certainly
happen in the cell.
What is bound water?
From the physicists point of view, then, the behaviour of water around solutes and in
confined geometries is full of complexity. Experience with simple liquids tells us that in
such situations a liquid deviates from bulk-like behaviour; the question of what waters
hydrogen-bonded structure adds to this picture is still open in many ways.
But biologists have tended to gloss over these details by invoking a simpler picture of cell
water. For a long time they distinguished two quite different kinds of water molecule:
bound and free. Free water was assumed to behave much as it does in the bulk. Bound
water is hydrogen-bonded, or at least quite firmly attached, to proteins and other
macromolecules, so that it remains in place when the macromolecule is crystallized.
Bound water constitutes 30-50% of the mass of dry protein powders, and up to 80% of
the mass of crystalline DNA. These water molecules are like limpets attached to the

macromolecule, and their positions are generally related to the proteins own atomic
structure, so that the waters can become visible in X-ray crystallography and neutronscattering experiments on protein crystals.
[slide] Yellow, green and purple spheres show bound water in different hydration shells.
This bound water can thus be considered an integral part of the biomolecule itself. There
are now many examples of this water playing an essential role in protein function, and I
want to give you a few examples of this.
Water wires:
Water trapped as hydrogen-bonded chains in protein channels seems able to act as a kind
of proton wire, transferring protons rapidly via the Grotthuss process of hydrogen-bond
flipping.
[Slide]
This can facilitate the delivery of protons to a catalytically active site, for example in
cytochromes and bacteriorhodopsin. There is a hydrogen-bonded network of bound water
molecules inside the channel of bacteriorhodopsin (a bundle of seven transmembrane
helices), stabilized by acidic and basic residues on the helices. This channel acts as a wire
to conduct protons from a Schiff base group on the photosensitive retinal pigment, via a
H5O2+ group in which a proton is delocalized. [Rammelsberg et al., Biochemistry 37,
5001 (1998); Spassov et al., J. Mol. Biol. 312, 203 (2001) Klaus Gerwerts group at
Bochum.]
Transient proton wires have been implicated in the leakage of protons across cell
membranes. Such chains may be very short-lived, but can nevertheless transport protins
during this time because the Grotthuss mechanism can be so rapid. The very recent
simulation studies of water molecues inside the hydrophobic channels of carbon
nanotubes may provide a good model system for understanding hydrogen-bonded chains
in proteins. (Another analogue system was reported by Terao et al., JACS 123, 1046810474 (2001).)
Selective binding:
Quiocho et al. at the Howard Hughes Medical Institute in Texas have shown how water
in the binding pocket of bacterial arabinose-binding protein provides a part of the jigsaw
that enables the protein to bind its ligand L-arabinose selectively. [Nature 340, 404
(1989).] In a more recent study of carbohydrate binding by proteins, Geert-Jan Boons
from the Complex Carbohydrate Research Center in Athens, Georgia, and coworkers
have looked at the interaction between trisaccharides and the legume lectin protein
concanavalin A. In the natural complex with this trimannoside, there is a
crystallographically defined water molecule in the binding site which provides a
hydrogen-bonding bridge with the ligand. Perhaps surprisingly, this complex turns out to
have a higher binding constant than one with a trimannoside derivative in which there is
an extra hydroxy group which displaces the bound water. This seems to be because the
bound water supplies an enthalphic contribution to the binding energy that outweighs the
favourable entropy gain when the water is displaced. In other words, the bound water is
not just some accident of design, but is part of the fine tuning of the process a fine-

tuning that involves a really quite delicate balance between the entropic and hydrogenbonding contributions of bound water and water in the bulk-like liquid network. [JACS
123, 12238 (2001)]
Enzyme activity:
Bound water molecules can take part in chemical reactions in the active sites of proteins.
For example, it seems that a water molecule can act as a nucleophile to initiate splitting of
a lactam ring in the bacterial enzyme zinc lactamase, which is involved in antibiotic
resistance (lactams are used as antibiotics). [M. Krauss et al., J. Phys. Chem. B 105, 8040
(2001). Krauss is at the Maryland Biotechnology Institute.]
And Minoru Sakurai and colleagues in Tokyo (Inst. Technol.) have shown that hydration
of an enzyme (ribonuclease T1) tunes its electronic structure, relative to the gas phase, so
as to make the frontier orbitals (HOMO and LUMO) those that are localized within the
enzymes active site. In other words, the enzyme is designed for use in water. [K. Ohno et
al., JACS 123, 8161 (2001).]
Allosteric effects:
William Royer et al. (Univ. Mass Medical Centrer) have reported evidence that water
molecules bound at the interface of the dimeric haemoglobin of the mollusc Scapharca
act as transmission units for allosteric effects that promote the cooperativity of oxygen
binding. In other words, structural changes in one subunit on binding an oxygen molecule
are communicated to the other subunit to increase its oxygen affinity via interfacial water
molecules between the subunits.
DNA:
Four hydration shells around DNA are visible to X-ray crystallography. This water sheath
becomes part of the structure, and it seems that these layers can act as a kind of switch
that, by flipping hydrogen bonds, allows a ligand that binds in the major groove to release
bound sodium ions from the minor groove. Changes in the hydration shell can also act
allosterically to control ligand binding to DNA by affecting the stability of the doublehelical secondary structure.
What are the consequences of these subtle sensitivities of cell components to water
activity and structure? How is cell water tied up with the health of the cell? There is
some evidence that the nature of cell water differs in healthy and diseased cells. NMR
measurements of water mobility can be hard to interpret because they can in some sense
represent an average between free and bound water, which exchange rapidly on the
NMR timescale. But there is some indication that the mobility of cell water is generally
different from that of bulk water, and that these motions become more bulk-like in cancer
cells. Thus it has been claimed that life tames cell water, bringing it into a more highly
structured state (whatever that might mean exactly) that is better able to facilitate cell
molecular processes.
In any event, a clear picture emerges in which water is not lifes matrix in the sense of
being a passive backdrop on which the tapestry of life unfolds. Rather, it is a biomolecule
in its own right, albeit an extremely complex one that can engage in many different

processes and form almost a kind of versatile loose macromolecule that interacts with
proteins, nucleic acids and membranes.
This image casts a new light on the question of whether water is essential for life. No
other small solvent molecule has the right geometry and chemistry to form the kind of
extended, three-dimensional hydrogen-bonded network we find in liquid water. If this
structure is indeed crucial for enacting lifes delicate molecular processes, that
strengthens the case that lifeall lifedepends on water. If we assume that all chemical
processes complex enough to qualify as life will require the same kind of characteristics
we see in protein interactionsexquisitely crafted and self-assembling architectures,
molecular recognition, allostery, a combination of flexibility and robustnessthen it is
hard to identify any other solvent that seems likely to support such things. (True, some
enzymes can function in non-aqueous solvents, but only after folding in water.)
There is a very nice short article by Chris Chyba in The Planetary Report (May/June
1998 slide) in which he asks the question: why water? Chyba points out that the
habitable zone in solar systems is generally taken to be the range of distances from the
central star that can support liquid water. We have to be careful about this. Strictly
speaking, Europa lies outside our solar systems habitable zone but nevertheless seems to
harbour a liquid ocean because of tidal heating from Jupiter. The same may be true of
Jupiters other moon Callisto. And John Brownlee and others have pointed out that there
is more to a habitable zone than liquid water, so that habitability may be a more rare
phenomenon in the universe than one might naively expect.
All the same, we tend to delimit habitability first and foremost by following the water,
as NASA puts it:
(Water Lifes Elixir in the Solar System; the science strategy for Mars exploration is
"to follow the water"NASA web site.)
Chyba warns that we should beware the hydrocentricthat we should be wary of some
arguments for waters indispensability for life. Its hard to see how nonpolar solvents like
the liquid hydrocarbons thought possibly to exist on Saturns moon Titan could possibly
support complex chemistry, but Chyba does not rule out ammonia as a potentially lifesupporting solvent. I think, for the reasons given above, as well as others such as
ammonias much weaker amphoteric nature, it is unlikely that ammonia could replace
water as another matrix of life. One interesting question is how, if life were to have
evolved elsewhere, it has solved the problems that water poses, such as its tendency to
hydrolyse nucleic acids and proteins.
Where do we stand in terms of getting answers to these questions?
Mars probes:
Mars Odyssey: was launched last April, is now in planetary orbit and will begin mapping
in February. It will map the distribution of the elements and minerals on the planets
surface, especially water ice. It uses thermal emission imaging and gamma-ray
spectroscopy to identify elements.

Mars Express: scheduled for launch in early 2003, to arrive December 2003. The
orbiting spacecraft will search for signs of subsurface water, and the mission includes a
landerBeagle 2which will conduct exobiological and geochemical studies of the
surface.
Sample returns are scheduled for missions in 2014 and 2016.
Europa probes:
Europa Orbiter: scheduled for launch in 2008, to arrive in 2010. This will investigate
the putative subsurface ocean in more detail than the Galileo mission, and will search for
candidate sites for future lander missions.
I am placing my hopes on the Europa missions, for the simple reason that they can
potentially answer the key question about liquid water: does its existence on another
world simply make life possible, or inevitable?