Chapter 4. Biochemical Insights into Minerals
K
ey :
Knowing the biochemistry of minerals allows visualization of minerals in a biological
setting. Macro- and microminerals are present in all organs and bodily fluids. Some organs
tend to concentrate minerals, such as the thyroid gland amassing iodine. Many “biological”
minerals exist as complexes with proteins and other macromolecules as noted earlier. Thus,
we see minerals bound to enzymes, to cell membranes, to nucleic acids, etc. Those existing in
free ionic form are basically hydrated ions. In this chapter we will key on some of the more
important mineral complexes and their biochemical functions. This information goes hand in
hand with knowing the full scope of functions of minerals in living systems.
O
bjectives:
1. To examine the biominerals as they exist within the organism,
2. To identify complexes of minerals with proteins and other macromolecules,
3. To take the first steps toward understanding function,
4. To relate a mineral’s function to its structure, its ionic state, and cellular location.
I. The fundamentals
Minerals are everywhere… in organs, in tissues, in every cell of the body. The blood is a
rich source of sodium ions and chloride ions, the cytosol is filled with potassium ions, bone is
structured around calcium and phosphorous, red blood cells are rich in iron, and the nucleus is
filled with zinc-bound proteins. This brief scenario captures the omnipresence of minerals in
living systems. One must pause to realize the potential chaos that such a picture presents.
With minerals everywhere, what determines order, specificity or function? The answer to that
question is the biochemical form. It is form that determines function because no two dissimilar
minerals can exist in the same biochemical form
A quick overview of mineral functions is shown in Table 4.1. Biochemists characterize life
as a series of well-regulated pathways designed to keep the organism’s status quo.
Nutritionists see this as keeping the organism tuned to its immediate environment. A large
part of the maintenance is based on channeling energy to and from cells. Energy-rich and
nutrient-rich molecules in the food provide the sustenance of life. Although minerals cannot be
considered as an energy source, they can and do assist cells in extracting and preserving energy
from compounds in the diet. The series of internal chemical changes is part of metabolism, or
more specifically catabolism when the focus is on the destructive processes. Fundamental
principles of chemistry tell us that a compound will yield energy only when it is changed
chemically. The liberated energy or “free energy” can be exploited to drive energy-demanding
reactions. Higher animals require oxygen to fully extract all the energy in food molecules.
While its beyond our score to revisit each step in metabolism, it will be our aim to learn the role
of minerals in the overall process of metabolism. Table 3.1 gives this overview.
Na+,
K+,
Cl-
Osmotic control
Electrolyte equilibrium
Ion currents
Gated channels
Mg2+
Phosphate metabolism
Ca2+
Muscle contraction
Cell signaling
Enzyme cofactor
Blood clotting
Mineralization
Morphogenesis
Gene regulation
Se
Redox reactions
Antioxidant
Mb2+
Enzyme cofactor
Nitrogen activator
HPO4=,
Acid-base non metals
Biomineralization
Si
Co3+
Lewis acid
Enzyme cofactor
Protein structure
Hormone activator
Neurotransmitter
Genetic expression regulator
Zn2+
Fe2+,
Fe3+
Heme iron
Electron transport
Oxygen activator
Oxygen carrier
Cu+, Cu2+
Enzyme cofactor
Oxygen carrier
Oxygen activator
Iron metabolism
Cr3+
Insulin mimetic
Glucose metabolism
Mn2+
Enzyme cofactor
Ni2+
Coenzyme
Remnant of early life
Vitamin b12
Table 4.1. Biochemical Functions of Select Minerals
2. Minerals Required to Metabolize Glucose
Figure 4.1 outlines the major set of reactions involved in extracting energy from glucose.
Highlighted are the minerals that come into play in each step in the process.
Glucose
Hemoglobin
Fe2+
Na+
intestine
mitochondria
O2
Glucose
H2O
liver
Mg2+
Mn2+
K+
Ca2+
PO43-
PO43Fe2+
Mg2+
Cu2+
Figure 3.1. Major minerals that take part in the metabolism of glucose
Note the absorption of glucose by intestinal cells is strongly dependent on sodium ions. to
drive the movement of the glucose-transporter complex inward. Upon entering the blood
glucose is absorbed by the liver where it is catabolized by a series of enzymes that require
Mg2+, K+ and Mn2+ as cofactors. A phosphate group from ATP is attached to the glucose via a
kinase enzyme that requires Mg2+ as a cofactor. Attaching the phosphate group serves two
purposes: (1) to keep the glucose trapped in the cell and, (2) to allow enzymes further down
the pathway to recognize and act on the glucose-phosphate complex. The final step in the first
phase is for the glucose, now converted to 2, 3-carbon phosphate units to enter the
mitochondria for further processing. The enzyme, pyruvate carboxylase, a Mn2+- requiring
enzyme, is important in one entrance pathway. A second goes via acetyl-CoA which is
converted into citrate by a zinc-dependent enzyme. In the mitochondria the final stages of
energy extraction take place. Hemoglobin, which is rich in Fe2+, brings oxygen into the cell to
drive the oxidation and Fe2+ and Cu2+ within the electron chain complete the transfer of
electrons to the oxygen forming water. A byproduct of all of these steps is ATP which
preserves the energy from the extraction steps. Adding up all the steps it can be seen that at
least eight different minerals and complexes play a major role in obtaining the energy from
glucose.
3. Minerals as Cofactors for Enzymes
The sight-unseen minerals alluded to in Figure 4.1 are mostly associated with enzymes.
Literally one-third of all enzymes in biological systems require a metal ion cofactor. The
indispensability of a mineral cofactor can be traced to the number of functions the metal ion
performs. These are (1) stabilizing the structure of the protein, (2) assisting the substrate in
binding to the active site of the enzyme, or (3) preventing electrons removed from a substrate
from contacting the delicate structure of the protein. As we look at the large category of
enzymes we note a need to distinguish whether the metal ion is in equilibrium with the enzyme
or is firmly attached to its structure. The former are referred to as “metal-activated” and the
latter as “metalloenzymes”.
3.1 Metal-Activated Enzymes: By definition, a metal-activated enzyme requires the metal
ion to be in the vicinity of the enzyme for maximum catalytic effectiveness. The metal ion does
not form a tight complex with the enzyme, but instead exists in a state of equilibrium. Upon
removal of the metal from the solution, the enzyme loses its activity. Adding back the metal
ion restores activity. In essence, a metal activated enzyme has a weak association of the metal
ion with the enzyme and functions optimally only when the metal ion is in the immediate
vicinity of the enzyme.
3.2 Metalloenzymes: Metalloenzymes, in contrast, bind the metal strongly to the surface
of the enzyme protein, making the metal an integral part of the structure. In many
metalloenzymes the metal ion is within the enzyme’s active site. Because of the tight binding,
there can be no equilibrium between free ion and enzyme. As a metal-protein complex, the
number of metal ions per protein must always be an integral number. Adding more metal ion
to the complex will not improve the catalysis since the site are filled. Isolated metalloenzymes
retain their function despite being removed from the biological milieu, which bespeaks of the
strong bond of metal to protein. Some metalloenzyme have multiple metals in their structure.
Ceruloplasmin, a protein that oxidizes Fe2+ to Fe3+, for example, has as many as seven copper
atoms bound. Only rarely are there two or more different metal ions within the same enzyme.
Perhaps the most familiar example is the enzyme superoxide dismutase which has two
subunits, each containing one atoms of copper and atom of zinc, the copper for catalysis and
the zinc for structural stability.
One of most intriguing aspect of metal-activated vs metalloenzymes is in the nature of
the metal ion involved. For metal-activated enzymes the metal generally belongs to the
macro-mineral category such as Na+, K+, Mg2+. In contrast the metals in metalloenzymes
constitute transition elements such as Mn2+, Fe2+, Cu2+ and Zn2+. Indeed, zinc alone has been
found in the structure of over 300 enzymes. Ca2+ is unique because it straddles the boundary
between metal-activated and metalloenzymes. Some hydrolase enzymes (those that break
bonds by adding water across the bond) require Ca2+ in the medium for maximum function,
whereas in thermolysin the Ca2+ is bound strongly to the enzyme’s structure.
Zinc (over 300)
Dehydrogenases
RNA, DNA polymerase
Carbonic anhydrase
Carboxypeptidase
Amino peptidase
Manganese
Arginase
Water splitting enzyme
Pyruvate carboxylase
Cobalt (with B12)
Copper
Superoxide dismutase
Tyrosinase
Cytochrome oxidase (with Fe)
Lysyl oxidase
Peptide amidating
Dopamine beta hydroxylase
Methylmalonyl CoA mutase
Homocysteine transmethylase
Molybdenum
Nitrogenase
Xanthine oxidase
Calcium
Iron
Thermolysin
Ribonucleotide reductase
Cytochrome oxidase (with Cu)
Nickel
Urease
Table4.2. Examples of Metalloenzymes
3.3 Metalloproteins: By definition, metalloproteins are a broader class of metal-binding
proteins that have no perceived catalytic activity. More often metalloproteins are used to store
metal ions or transport them in the blood and within cells. Their role in storage is linked to
detoxification, which is basically removing the metal to prevent toxicity. The capacity of
metalloproteins to select specific metal ions gives insight into their uniqueness. The protein
ferritin, for example, has been found to hold between 2,500-5,000 atoms of iron in one protein
molecule. That number could go higher depending on circumstances. Another protein,
metallothionein, binds both copper and zinc and can also engage cadmium ions. Transferrin,
another iron binding protein binds and transports two atoms of iron to different organs. The
main function of transferrin, therefore, is metal ion transport. Albumin in the plasma also binds
and transports metals mainly zinc and copper. Thus metalloproteins have a varied but highly
important role in maintaining cells in a healthy state and keeping physiological systems safe
from the toxic action of metal ions. Table 4.4 is a partial list of metalloproteins and their
function.
Protein
Function
1. Metallothionein
Cu, Zn, Cd storage, heavy metal buffer
2. Ferritin
Iron storage, iron buffer
3. Calmodulin
Ca binding, allosteric regulator
4. Transferrin
Iron transport
5. Selenoprotein W
Selenium transport
6. Calbindin
Calcium transport
7. Serum albumin
General metal ion transport
8. 2-macroglobulin
Zinc transport
Table 3.4. Examples of metalloproteins and their function
4. Biomineralization
Combing calcium with phosphorous (as phosphate) and calcium with carbon (as
carbonate) are two examples of bulk minerals being deposited for the purpose of forming
major supporting structures. Bone, teeth and egg shells represent two such examples in higher
animals. This unique happening should not go unnoticed. Bone is brought about by combining
one forming units referred to as hydroxyapatite. The crystallization process occurs
spontaneously and requires osteobasts and osteoclasts, two special bone forming and
remodeling cells.
Egg shell formation requires a special organ, the shell gland. Mineralization begins with
the deposit of calcium carbonate around a fibrous protein layer that encompasses the yolk and
ovalbumin and it passes down a tube. Placing the crystalline coat is the final stage and involves
the same quantity of calcium and carbonate committed to the shell, regardless of the size of
the yolk and ovalbumin core. Large diameter cores, therefore, could result in thin-shell or even
no-shell eggs for that reason. For the consumer, this manifest as large and extra-large size
eggs caused by a poorly managed mineralization process that cannot adjust to egg size.
5. Zinc as a cofactor for Enzymes and Proteins
Later chapters will discuss the nutrition of individual minerals. In discussing their
biochemistry here our goal is to learn their biochemical structures as this relates to their
function. Zinc is one such mineral with a multitude of functions. More than 300 enzymes
require zinc as a cofactor. Its role in these enzymes is varied. One reason for its versatility is
that is zinc’s ability to link to amino acid side chains in protein. As we learned in Chapter 2, the
Hydroxyapatite (crystal structure)
Ca10(PO4)6 OH2
Ca
P
O
H
Figure 3.2. Biochemical structure of bone
binding of zinc must adhere to a specific geometry at the binding site as shown in Figure 4.2.
Figure 4.2 Zinc as a structural component of enzymes
Only ions that can duplicate the stereochemical orientation of the bonds will be able to bind at
the site. Thus, even though the two plus charges on Zn2+ suggests cationic character, zinc does
not bind to proteins through a charge-charge interaction but instead by coordinate-covalent
bonds with specific geometric constraints (Chapter 2). Zinc bonding in carbonic anhydrase is
basically tetrahedral with three histidine groups engaging directly and one open valence for
water. When the water binds to the zinc, one of its protons is lost and it behaves as OH-, which
is a stronger nucleophile than water. This is shown in Figure 3.3. Note how the water
molecule is activated by the zinc ion an attack on the CO2 eventually forming a bicarbonate ion,
a quintessential reaction for converting CO2 gas to an ion. More important, however, is that
the reverse of this reaction in the lungs changes bicarbonate to CO2 which is exhaled as a gas.
His
His –Zn2+
His
His
O
..
O
+ C
H
O
O
His –Zn2+
O
C
O
H
His
H2 O
His
His
–Zn2+
His
-
Displaces HCO3
..
O
H
+
H+
O
+
H O
C
O
Bicarbonate
Figure 3.3. Catalytic action of zinc in carbonic anhydrase
Another important function of zinc is the control of genetic expression. Figure 3.4
shows zinc as a complex with proteins that regulate transcription of DNA (mRNA synthesis).
Without zinc, the transcription protein cannot bind to the DNA. Zinc’s role here is basically
structural in that a loop is formed in the amino acid chain giving the appearance of a “finger”
and hence zinc-binding transcription factors are referred to as “zinc-finger proteins”.
S
N
Zn
S
N
Figure 3.4. Requirement for zinc in a “zinc-finger” transcription factor. Shown
are two cysteine -SH groups (green) and two -N histidine imidazole groups
(blue) engaging the zinc.
6. Biochemical Forms of Iron
When we consider iron in a biological system, we must pay heed to a metal that has the
potential to cause harm to a cell. Iron is bound strongly to proteins for that reason. Part of
this is due to Iron’s redox character which renders it as a pro-oxidant capable of donating and
receiving electrons and in the process generating free radicals. The entrance of iron into the
biosphere was timed with the enrichment of atmospheric oxygen, itself a dangerous gas.
Iron’s role was to assure the safe utilization oxygen. Iron, however, also exists in multiple
valence states, Fe2+ and Fe3+, giving it redox character. Changes in valence can be exploited in
the mitochondria where iron receives and donates electrons as part of an electron transport
chain, oxygen being the final recipient. The strange paradox is that iron both binds oxygen
and takes part in its reduction to water, two seemingly opposing functions.
Unlike zinc which has a single +2 valence and limited biochemical forms, iron is present
in a multitude of forms and valences. Most of the iron in the system exists as a complex with
porphyrin giving rise to heme (Fig. 3.5). Its other major form is that of an iron sulfur center in a
protein. Both are found in the core, not the surface, of a protein and both forms occur in the
Iron-Sulfur Centers
Heme Iron
Figure 3.5. Major biochemical forms of Iron
mitochondria, where iron’s major role is the transport of electrons to oxygen as part of
respiration (oxygen uptake). Iron as heme is found in hemoglobin and myoglobin, two proteins
designed to transfer oxygen. By binding to the porphyrin ring, iron makes only a single contact
with the protein’s amino acids. Iron-sulfur centers, in contract, require extensive interactions
with the sulfur groups of the protein, primarily cysteine –SH groups. Forming an iron-sulfur
center is more suited to transferring electrons and is not to binding oxygen. Thus, moving
electrons and transferring oxygen, the two major function of iron in biological systems, are
possible because of two distinct biochemical complexes or proteins with iron.
7. Biochemical Forms of Copper
Copper’s advent into a biological system was also in response to an enrichment in
atmospheric oxygen. Although copper as a transporter of oxygen occurs in mollusks and
therefore, mimics iron in a non-vertebrate, copper’s primary use is as a cofactor for
“oxidases”or enzymes that use molecular oxygen as a substrate to accept electrons. Copper
oxidases are quite common in the plant and animal systems. Based on the number of copper
atoms bound, copper oxidases are further subclassified as mono- and multi-copper oxidases,
referring one or more than one copper atom in the protein, respectively. The number of
copper atoms is an important consideration because the product of an oxidase will either be
hydrogen peroxide or water, depending on the number of electrons transferred. For example,
multi-copper oxidases give rise to water as shown in the formula (a). This occurs when four
electrons are transferred to oxygen from a substrate (AH2). Hydrogen peroxide is formed
when only two electrons are transferred as shown in (b).
(a)
(b)
AH2 + O2 + 4e
AH2 + O2 + 2e
2H2O + Aox
H2O2 + Aox
Having four copper atoms in the protein allows for a coordinate delivery of electrons to
the oxygen. Two of the copper atoms take part in binding the oxygen whereas the others are
part of an electron delivery chain. Perhaps the most notable feature of multi-copper oxidases
is the “blue” copper center in the protein. This center gives the protein a decidedly blue tint
that becomes very apparent when the protein is isolated in pure form as shown in the figure
below for the protein ceruloplasmin.
Purified Plasma
Ceruloplasmin
Copper Centers in Cu Oxidases
Blue Copper (Type 1) Center
and
Trinuclear (Type 2 + Type 3)
Figure 3. 6. Copper centers in multi-copper oxidases. Shown is the plasma protein
ceruloplasmin with its blue color caused by a unique arrangement of amino acids.
7.1 Ceruloplasmin One of the most important copper proteins in mammalian plasma is
called ceruloplasmin. The name means “heavenly blue” plasma protein. As seen in Figure 3.6,
ceruloplasmin has an intense blue color caused by the presence of a specific coordination of
copper to amino acids making up what is called the “blue copper center” of the protein.
Ceruloplasmin is also called ferroxidase denoting its ability to catalyze the oxidation of Fe2+ to
Fe3+. Only Fe3+ is capable of binding to transferrin, the iron transport protein. That reaction is
needed for the system to use the iron taken in the diet.
7.2 Superoxide dismutase Since ionic copper is capable of taking and giving electrons, it
is perfectly within the properties of copper to be at the active site of an enzyme that intercepts
free radicals. In so doing copper’s other important role is that of an antioxidant. Its
antioxidant activity is linked to the enzyme superoxide dismutase. Superoxide dismutase
destroys superoxide anion (O2-), an oxygen radical. The products of the reaction are molecular
oxygen and hydrogen peroxide as shown below. This is a two-step reaction with copper taking
part at each step. In the first step the single electron in O2- is ensconced by the Cu2+ forming O2
and reducing Cu2+ to Cu+. In the second step Cu+ transfers the electron to a second O2- causing
the formation of hydrogen peroxide as the second product and restoring Cu2+. The reaction is
shown below.
O2- +
Cu2+
O2- + 2H+ + Cu+
O2
+ Cu+
H2O2 + Cu2+
2O2-
O2 + H2O2
+ 2H+
A second cofactor for the enzyme is zinc. Zinc, however, plays mainly a structural role and does
not take part in the electron exchange. Note that copper recycles through both valence states
to achieve the desired outcome.
4. Selenium and Iodine, Notable Exceptions
Although the foregoing discussion applies to most minerals, there are some notable
exceptions. Prominent on the list of exceptions are selenium and iodine . Neither are metals
nor organic components, but both engage organic components by covalent bonds.
Technically, selenium is referred to as a metalloid inferring its existence is somewhere in
between covalent and ionic forms.
Selenium belongs in the same class as sulfur and thus can exist in multiple valence states
with redox properties. It breaks the rule, however, in being a component of amino acids, two
of which are selenomethionine and selenocysteine (Fig. 2.1). As a component of the structure
of an amino acid, selenium can easily be brought into the structure of a protein. Visualize
selenium within the structure, not as an appendage attached to amino acids. This remarkable
H
HSe-CH2
H
-C-COO-
CH3 -Se-CH2CH2 C-COONH3 +
NH3 +
Figure 4.1. Selenocysteine (left) and Selenomethionine (right)
substitution of a sulfur atom for a selenium atom gives rise to enzymes that perform
antioxidant functions, functions that cannot be supported if the selenium atom is replaced by a
sulfur atom.
Iodine is unique is forming a covalent complex with an organic molecule giving rise to
family of hormones referred to thryroxins.
8. Iodine (iodide ion)
The last mineral to consider for unique biochemical forms is iodine. Iodine (or iodide
ion) is part of group of halogens (halides) which includes chlorine (chloride ion) as its most
I
HO
I
O
CH2 CH COOH
I
I
NH2
T4
I
HO
I
O
CH2 CH COOH
I
NH2
T3
prominent biological member. Unlike its fellow halide, iodide ion does not exist in free form in
a biological system. Rather iodide is bound to the ring system that makes up two forms of the
thyroid hormone, T3 and T4 (Fig. 3.7). The numerical component refers to the number of iodine
atoms bound.
9. Summary
When we view a mineral in its biological setting it becomes apparent that minerals are
present in a multitude of biochemical forms. Very few minerals exist as free ions in solution.
One reason is free ions have limited biochemical capabilities. Minerals amass to the greatest
extent in the calcium-phosphate matrix of bone and the calcium carbonate coat of egg shells.
A large group is bound to proteins and most of these minerals function as enzyme cofactors.
Up to one-third of all enzymes require a metal ion to function optimally. Minerals contribute to
both the structural and catalytic properties of enzyme. Two categories of mineral-dependent
enzymes are “metal-activated” and “metalloenzymes”. The two differ in the strength of metal
binding, whether in equilibrium or firmly fixed to the structure. Metal ions, particularly those
with redox properties, tend to be bound to enzymes that remove or add electrons to
substrates. Iron is unique in being a metal that can both reduce oxygen to water or carry
oxygen to tissues. The two functions occur because of two distinctly different biochemical
forms. Zinc is prominent in many enzymes and plays an important role in genetic expression.
Copper enzymes use molecular oxygen as a substrate. Finally, iodine is an important
component of thyroid hormones. The hormones cannot function if iodine is missing from their
structure. The biochemical properties of minerals reveal a multitude of different structures.
On closer inspection it becomes apparent that the complex of the mineral goes hand in hand
with the function performed. Some of these, nonetheless, still remain a mystery, such as why
thyroid hormones need to have iodine attached in order to be active.
10. Problems to Ponder
1. Obtain a biochemical text book to answer the following.
a. What role do chloride ions play in the absorption of glucose
b. A kinase enzyme uses ATP as a substrate. What mineral is needed for the
enzyme to function?
c. The enzyme pyruvate carboxylase uses a mineral as a cofactor. Name it.
d. Where is iron found in the electron transport system in mitochondria. How
many different forms of iron are present. Where is copper found in
mitochondria?
e. What function is performed by the enzyme Na+/K+ ATPase.
2. Describe the structure of heme. What is the valence state of the iron in heme?
3. What amino acids make up a blue-copper center in ceruloplasmin? How is the copper
bound to them?
4. Where do you find hydroxyapatite in a biological system? What is hydroxyapatite?