ARTICULO 1

Principles in Bioinorganic Chemistry

The biochemical roles played by essential inorganic elements and compounds are:
1) structural
2) carry ligand transporting electrons and oxygen
3) catalytic roles in oxidation-reduction reactions, and
4) catalytic roles in acid-base and other reactions

Many inorganic elements and their compounds are now known to be essential to organisms (1). Organic
compounds are of course essential, because they provide organisms with such essential compounds as
proteins, nucleotides, carbohydrates, vitamins, and so forth. Inorganic compounds, particularly metallic
ions and complexes, are essential cofactors in a variety of enzymes and proteins. They conceivably
provide essential services which can not be or can only poor be rendered by organic compounds. The
roles played by essential inorganic elements and compounds are (1) structural (2) carrying and
transporting electrons and oxygen, (3) catalytic roles in oxidation-reduction (including oxygenation)
reactions and (4) catalytic roles in acid-base and other reactions. The type of question we are interested
in here is why a certain in- organic element (compound) is specifically required for a certain function in
biological systems. For example, why is cobalt uniquely required for vitamin B12 and the enzymatic
reactions dependent on B12? Why not iron or copper? We think that questions of this type may be
answered at least partially in terms of basic inorganic chemistry. This is what this article attempts to
show. First let us lay out some basic principles regarding the above question. They are:

1) Rule of abundance
2) Rule of efficiency
3) Rule of basic fitness
4) Evolutionary improvement of efficiency and specificity
We will illustrate these principles by a few examples.

Rule of Abundance
The rule of abundance may be stated as: When a function can be accomplished by two or more entities;
organisms would utilize the more abundant, readily available one. Examples of this rule are numerous.
As a general rule, the lighter elements are more abundant. The elements essential to organ- isms are
mostly light elements, lighter than atomic number 34 (selenium), with exceptions of iodine and
molybdenum. Molybdenum, as MoO4(2-), is present in a rather high concentration, as high as iron in sea
water. The concentration of zinc in sea water is also on the same order of magnitude as that of iron; the
copper concentration is about half as high. These four elements are the most frequently found in the
catalytic sites of enzymes. The use of the most abundant alkali metals, sodium and potassium, in
controlling ion balance and enzyme activities is also in accord with this rule.

Most organisms utilize calcium compounds such as carbonate and phosphate as protective and skeletal
material. Undoubtedly this is due to the insolubility of calcium carbonate and phosphate. However, the
corresponding strontium compounds are equally insoluble and could substitute calcium compounds. It is
obvious that calcium is much more abundant than strontium (1,2). An interesting exception is a group of
marine protist, radioralian; their outer skeletons are made of either strontium sulfate or silica. Somehow
they could not develop a mechanism to utilize more abundant calcium or their specific need may not be
satisfied by calcium com pounds.

Zinc (2) in zinc-enzymes can be in most cases replaced by cobalt (2) in vitro without losing catalytic
activity (3). If organisms are grown in cobalt-rich media, they can produce enzymes in which Zn (2) is
replaced by Co(2). For example. . . when Escherichia coli is in the presence of relatively high
concentration of 60 Co(2) .a catalytically active 60Co (2) alkaline phosphatase is obtained (4): thus, zinc
and cobalt seem to be interchangeable, but organisms selected zinc because zinc is much more
abundant both in sea and earth's upper crust.

A general discussion of the relationship between the abundance of elements and their essentiality or
toxicity was made elsewhere (1,2).

Rule of Efficiency

The rule of efficiency asserts that organisms would choose the more efficient entity as long as it is
readily available. Flavodoxins (5) and ferredoxins function as electron-carriers in very similar ways, being
interchangeable in most cases. However. their compositions are entirely different. Flavodoxin contains
flavin mononucleotide (FMN) as the prosthetic group, whereas the functional units in ferredoxins are
ironsulfur complexes. Flavodoxin in general is less efficient than ferredoxin. and the synthesis of
flavodoxin occurs only during growth iron-poor media in a number of microorganisms. for example. in
the case of peptostreptococuus elsdenii, iron-rich cells were found not to contain flavodoxin and iron-
deficiencies brought about its de novo synthesis (5).

Rule of Basic Fitness

Primitive (in historical sense) organisms tried to improve their survival chances by tapping whatever
resources were available to them. For example, they might have explored many transition metal
complexes in order to effect the oxidation-reduction reactions. As the results of this endeavor, they
selected iron. copper. and molybdenum and their complexes to effect their desired oxidation-reduction
reactions. This third basic principle might be stated as: an inorganic element (generally a metal) to be
selected should have a basic ability or potential to carry out the desired function. That is, a certain
element (or elements) would inherently fit to a particular function.

As mentioned earlier, most organisms utilize calcium compounds as protective material. An important
group of organisms, diatom, use silica as their outer cover, despite the fact that silicon is much less
abundant than calcium and strontium in sea water. Diatom is a kind of algae. There are a vast number of
different kinds of algae and all algae other than diatom do not have any cover of inorganic material.
though there are a few green and red algae, mostly extinct; which have slightly calcified walls. Somehow
the predecessor of diatom happened to try a coat of silica and found it very useful for its survival.
Perhaps some other algae might have tried limy coats but did not find them useful. Probably it is due to
the fact that silica forms transparent material whereas limy coats are often much less transparent.
Algae. of course. have to absorb sunlight to support their photosynthesis.

An element may have ranges of capacities modulated by external factors such as coordinating ligands.
However, these ranges are not limitless, and are more or less confined within certain values, particularly
when the kinds of external factors are limited. Some of the important restrictions in the case of ordinary
biological systems are: (1) the medium is water, (2) the range of temperature is rather narrow, and (3)
the possible coordinating ligands are limited; they are proteins, carbohydrates. nucleotides. lipids, and a
few other specific ligands such as porphyrins.

The catalytic effects of a metal ion can be characterized by many factors (6). They are: (1) continuous
parameters such as reduction potential and Lewis acidity and (2) discontinuous (discrete, parameters
such as number of valence electrons and favorable coordination number (structure). The parameters in
category (1) are modulated by ligands and other external factors such as pH of the medium, but those in
category (2) are much less influenced by ligands and others.

Let us take reduction potential as an example and illustrate how the rule of basic fitness may work.
Figure 1 shows the reduction potentials for several important metal ions and other redox systems. The
dotted lines represent the oxidation and reduction of water and the pH-dependency of the re duction
potentials. The points shown by (o) are the reduction potentials of the aquo species at (pH = 0 ) and
those of the corresponding hydroxo complexes at (pH = 14). The two points at (pH =0) and 14 are joined
by a straight line which does not necessarily represent the pH dependence of the reduction potential.
The reduction potential for a few ordinary complexes are shown on the extended broken lines. The
reduction potentials (at pH 7) of the metalloenzymes and metalloproteins containing copper and iron
are shown by (black point) o (white triangle) on the respective straight lines or the broken lines in Figure
2.

The following items are some of the points that we can infer from Figures 1 and 2.

1. Since the species whose reduction potentials lie outside of the decomposition lines of water
(dotted lines) would (in thermodynamic sense) either oxidize or reduce water, they may not be
suitable for a catalytic entity working in aqueous media. This consideration alone would
probably exclude Co(3)/Co(2), Sn(4)/Sn(2), and Cr(3)/Cr(2) systems as candidates for redox
catalysts (enzymes).
2. The entire range of the reduction potentials of a variety of iron-enzymes and proteins is
confined in the potential range of the iron-aquo system. The potentials of iron-sulfur
proteins are closer to that of the (Fe2)(3)/(2Fe)(2)S system. These facts may indicate
that the prebiotic catalysts for those reactions carried out today by iron-containing
enzymes and proteins might have been Fe(H20)6n (OH)n, and (FeS/Fe2S3) or their
simple complexes. These simple compounds were later incorporated into porphyrins
and proteins, leading to the formation of mare efficient and selective catalysts, the
potentials, however, did not change very widely. In other words, these simple
compounds that might have been readily available to precursors of organ- isms or
primitive organisms are basically fit to the specified requirements (chemical reactions). It
should be noted, however, that iron porphyrins themselves might have been the first
catalysts, since porphyrins are known to be one of the mast ancient compounds
synthesized abiotically (7).
3. The situation of copper species is slightly different from that of iron. Cuprous aquo
species are not stable and disproportionate into Cu(0) and Cu(2). This would preclude
the use of the simple aquo (Cu(2)/Cu(1)) system as an oxidation-reduction catalyst.
Instead, such simple complexes as (Cu(2)Cl2/CuCl2-Cu(2)), (pyridine)2Cu(1)(pyridine)(2),
or similar complexes could have provided the basis for prebiotic or primitive catalyst.
Cu(2)Cl(-1) species could indeed be predominant in a marine environment. The
 reduction potentials of these compounds are basically fit to those of present-day copper
enzyme and proteins.
4. The reduction potentials of (O2-/H202) and (02/02-) are +0.96 V and -0.45 V (at pH 7),
respectively. Suppose that the mechanism of superoxide dismutase reaction is simply as
follow. (see reaction), Then it would be inferred that a redox system whose potential lies
somewhere in the middle of the range +0.96 V to -0.45 V would function as a catalyst for
the superoxide dismutase reaction. Figure 1, indicates that such systems are Fe(3)/Fe(2),
Cu(2)/Cu(1), and Mn(3)/Mn(2). In fact, superoxide dismutase from different sources
contain copper, iron, or manganese.

(8) A superoxide dismutase containing copper was found to have a reduction potential of + 0.42 V
(9). As far as the reduction potential is concerned, the aquo 5(4)/5(3) system is also fit far
superoxide dismutase function. One of the functions of hemovanadin, which is a
vanadium-containing protein found in a marine invertebrate, ascidian, might well be a superoxide
dismutase.

Let us turn to another example, oxygen-carrying proteins (10). This function can be traslated into
the reversibility of the reaction to bind oxygen; that is. (see reaction 2).

In order for these reactions to be reversible, (AGa) or (AGb) should be roughly (-2) to (-12) kcal/mole
(9). If (AGa), (or AGb) menor a (-2) kcal/mole, the forward reaction occurs to only a small extent. On
the other hand. if (AGa). (AGb) menor (-13) kcal/mole. the reaction goes essentially to completion
and the reverse reaction occurs to a negligible extent. A rough estimate for ( AGa), and (AGb) was
made from reduction potentials and is given in the table (10). This table indicates: (1) the reversible
oxygenation of type (a) is very probable with some Fe(2) and Co(2) complexes and possibly some
Cr(2) complexes, and (2) the reversible oxygenation of type (b) may be carried out by some Fe(2),
Co(2), and Cu(1) complexes. This conclusion fits the known facts (9).

An example of the effect of discrete parameters seems to be vitamin B12, cobalamin.

The mechanisms of B12 coenzyme-dependent enzymatic reactions are still under intensive studv
and are far from settled (11. 12). However. the important; requirements for the candidates for the
job appear to be rather well defined. They are: (1) it should readily take three consecutive oxidation
states, for example, (1),(2) and (3) in aqueous media, (2) the lowest oxidation state should be
highly nucleophilic, and (3) the middle oxidation state should per- haps have one unpaired electron
(11). The requirement (2) implies that the lowest oxidation state of the catalytic metal ion should
have (d8) or (d10) configuration. This condition may be satisfied by Fe(O), Co(1), Ni(O), and Cu(1).
Fe(O), and Ni(0) are not readily attainable, particularly in aqueous media. This leaves us Co(1) and
Cu(1). Cu(1) is not particularly nucleophilic and Cu(3) is not readily obtained. Only a
Co(3)/Co(2)/Co(1) system satisfies all these requirements. This explains why cobalt (cobalamin)
uniquely fits the job (11).

Other applications of the rule of basic fitness to more specific cases will be discussed elsewhere.
Evolutionary Improvement of Efficiency and Specificity

The rule of basic fitness dictates which element (s1 is suit- able for a specific enzymatic reaction only
in terms of thermodynamics. The element or its simpler compounds selected maybe able to do the
required function but may not be very efficient in the senses of kinetics and specificity. The
substrate specificity and the efficiency seem to be controlled mainly by the protein portions of
metalloproteins or metalloenzymes. Thus, the improvement of these functions must have taken
place in early stages of evolution, involving the mutation in the genetic codes. We know little about
this, hut we may be able to rationalize some of the results from an inorganic standpoint. What we
are concerned with here is the efficiency of many enzymes. Their rates are usually very much higher
than those of simple model compounds. Vallee and Williams (13) proposed that the structure of the
active site of an enzyme, particularly a metalloenzyme, is rather distorted or strained and that it is
responsible for its high catalytic activity. They called it “Entatic Effect” Let us now translate this into
chemical language.

Very generally speaking, the rate is most dependent on the activation energy. The activation energy
can be defined in terms of absolute rate theory as the potential energy difference between the
initial and the transition state. that is. the activation energy (AG*), for the process (ML) + (S)=
(MLS)*, where ML is a metal complex and (S) is a substrate including electron. A significant portion
of (AG*) comes from the energy necessary to reorganize the structure of ML. so that ML. take the
structure in (MLS)*. Therefore, the closer the structure of the initial state ML. is to that of the
transition state, the lesser (AG)* becomes. A few examples will suffice to illustrate this point.

Blue copper proteins (14) such as plastocyanin and azurin function as electron-carriers, in which
copper oscillates between Cu(2) and Cu(1). The favorable coordination structure of Cu(2) is square
planar and that of Cu (1) is tetrahedral. The reduction of a square planar Cu(2), therefore, would
require a significant rearrangement in the structure about copper ion. One-way to reduce this cost is
to start with a compound whose structure is between the regular square planar and the regular
tetrahedral one. There is now convincing evidence that the coordination structure about copper
atom in the blue proteins is indeed distorted-tetrahedral (see (15) for review).

Other examples are zinc enzymes (16). The structural studies by X-ray crystallography and by
spectroscopy of the cobalt (11)-substituted enzymes have established that the coordination
structures about zinc atom in carbonic anhydrase, carboxypeptidase (A), and alkaline
phosphatase are distorted tetrahedra, being between a regular tetrahedron and a five-
coordinate trigonal bipyramid (3). The function of these enzymes is to hydrate the substrates.
Not only the substrate but also the water molecule would have to be activated. The distorted
tetrahedral structures of the enzymes usually have one water molecule and three amino acid
residues coordinated about the zinc ion. If the coordination structure about the zinc atom is
distorted from tetrahedron in such a way that the substrate can readily approach the zinc ion
(forming a five- coordinate bipyramidal type of structure), the whole reaction would be
facilitated. In a regular tetrahedron, this binding of an additional ligand would not be very easy.