Chem 554.pptx

BIOINORGANIC CHEMISTRY
Introduction
 Bioinorganic chemistry involves the study of metal species in
biological systems.
 It is devoted to all aspects of “inorganic elements” as being vital
for the growth and metabolism of living systems.
 Hence, the essential chemical elements, the occurrences and
purposes of metal centers in biological species, the geometries of
ligand fields surrounding these metal centers, and ionic states
preferred by the metals will be discussed.
1

 Organic chemistry at one time was thought to be the chemistry
involved in living systems because compounds of carbon were
found to be playing the key role in all biological processes.
 However, slowly it was realized that metal ions play a vital role in a
vast number of widely differing biological processes.
 Some of these processes are quite specific in their metal ion
requirements in that only certain metal ions, in specified oxidation
states can fulfill the necessary catalytic or structural requirements.
 A large number of metal ions are involved in biological processes.
2

 This can be verified by just going through the protein data banks e.g.
in Brookhaven Protein Data Bank 52% proteins contain metals
(excluding weakly bound metals such as sodium).
 This count includes proteins and enzymes with heme and corrin
groups and mutated forms of certain metallobiomolecules and
structures of the same enzyme with different substrates and
inhibitors.
 Metal ions play essential roles in about one third of enzymes.
 These metal ions can modify electron flow in a substrate or enzyme,
thus effectively controlling an enzyme-catalyzed reaction.
3

 They can serve to bind and orient substrate with respect to
functional groups in the active site, and they can provide a site for
redox activity if the metal has several valence states.
 Without the appropriate metal ion, a biochemical reaction catalyzed
by a particular metalloenzyme would proceed very slowly, if not
stopped at all.
 So bio-inorganic chemistry comes in to the play.
 The field is undergoing a phase of explosive growth, partly because
of exposure and insights obtained by large number of x-ray
structures of several metalloenzymes.
4

 It was only after 1960 that bioinorganic chemistry became an
independent and highly interdisciplinary research area.
 The following factors have been crucial for its development:
1. Biochemical isolation and purification procedures, such as
chromatography, and the new physical methods of trace element
analysis, such as atomic absorption or emission spectroscopy,
require ever smaller amounts of material.
 These methodical advances have made it possible not only to
detect but also to chemically and functionally characterize trace
elements or otherwise inconspicuous metal ions in biological
materials.
5

 An adult human being, for example, contains about 2 g of zinc in
ionic form (Zn2+).
2. Efforts to elucidate the mechanisms of organic, inorganic and
biochemical reactions have led to an early understanding of the
specific biological functions of some inorganic elements.
 Nowadays, many attempts are being made to mimic biochemical
reactivity through studies of the reactivity of model systems, low-
molecular-weight complexes or tailored metalloproteins.
3. The rapid progress in bioinorganic chemistry, an interdisciplinary
field of research, has been made possible through contributions from:
6

◦ physics (→techniques for detection and characterization);
◦ various areas of biology (→ supply of material and specific
modifications based on site-directed mutagenesis);
◦ agricultural and nutritional sciences (→effects of inorganic elements
and their mutual interdependence);
◦ pharmacology (→ interaction between drugs and endogeneous or
exogeneous inorganic substances);
◦ medicine (→imaging and other diagnostic aids, chemotherapy);
◦ toxicology and the environmental sciences (→ potential toxicity of
inorganic compounds, depending on the concentration).
7

 Bioinorganic chemistry encompasses a variety of disciplines,
ranging from inorganic chemistry and biochemistry to
spectroscopy, molecular biology, and medicine etc.
Figure 1, Bioinorganic chemistry as a highly interdisciplinary research
field
8

Essential Chemical Elements
 Essential elements are absolutely essential or necessary for life
processes.
 For an element to be considered essential, it must have a
specific role, where deficiency of that element results in
adverse affects that are reversed upon resupply.
 Essentiality has been defined by certain criteria:
(1) A physiological deficiency appears when the element is removed
from the diet;
(2) the deficiency is relieved by the addition of that element to the
diet; and
(3) a specific biological function is associated with the element. 9

 Trace elements are also necessary for life processes.
 Trace elements with regard to the human body involve a daily
requirement of less than 25 mg.
 Non-essential elements are not essential.
 If they are absent other elements may serve the same function.
 Toxic elements disturb the natural functions of the biological system.
10

 Every essential element follows a dose – response curve, shown in
Figure 2.
 At lowest dosages the organism does not survive, whereas in
deficiency regions the organism exists with less than optimal
function.
 After the concentration plateau of the optimal dosage region, higher
dosages cause toxic effects in the organism, eventually leading to
lethality.
 Specific daily requirements of essential elements may range from
microgram to gram quantities.
12

Figure 2, A dose – response curve for essential elements
13

How do inorganic compounds and ions help cause biochemical
reactions?
A partial list of their actions is given here, most related to metal ion
complexes.
1. Promotion of reactions by providing appropriate geometry for
breaking or forming bonds.
 Although many coordination sites in bioinorganic molecules are
approximately tetrahedral, octahedral, or square-planar, they have
subtle variations that provide for unusual reactions.
 An organic ligand may provide a pocket that is slightly too large or
small for a particular reactant, or may have angles that make other
sites on the metal more reactive.
14

 The binding of small molecules can also create reactive species by
forcing them to adopt unusual angles or bond distances.
2. Changes in acid-base activity. Water bound to a metal ion frequently is
more acidic than free water, and coordination to proteins enhances this
effect still more.
 This results in M-OH species that can then react with other substrates.
Mg2+ and Zn2+ are common metal ions that serve this function.
3. Changes in redox potentials. Coordination by different ligands
changes redox potentials, making some reactions easier and some
more difficult, and provides pathways for electron transfer.
15

4. Some ions (Na+, K+, Ca2+, C1-) act as specific charge carriers, with
concentration gradients maintained and modified by membrane ion
pumps and trigger mechanisms.
 Sudden changes in these concentration gradients are signals for nerve
or muscle action.
5. Organometallic reactions can create species that are otherwise not
attainable.
 Cobalamin enzymes are particular examples of these catalysts.
6. Inorganic ions, both cationic and anionic, are used as structural units
to form bone and other hard structures.
16

 Maintenance of cell membranes and DNA structure also depends on
the presence of cations to balance charges in the organic portions.
7. A few small molecules have specific effects that do not fit easily into
any of the categories above.
 Perhaps the most obvious is NO, which has many functions,
primarily related to control of blood flow, neurotransmission,
learning, memory, and, at higher concentrations, as a defensive
cytotoxin against tumor cells and pathogens.
17

2. Properties of Biological Molecules
 Biomolecules, also called biological molecules, are substances
produced by cells and living organisms.
 Biomolecules have a wide range of sizes and structures and perform
a vast array of functions.
 chemical compounds found in living organisms are of two types.
One, those which have molecular weights less than one thousand
dalton and are usually referred to as micromolecules or simply
biomolecules, while
 those which are found in the acid insoluble fraction are called
macromolecules or biomacromolecules.
18

 The four major types of macromolecules are carbohydrates, lipids,
nucleic acids, and proteins
 Carbohydrates: Carbohydrates are made up of sugar monomers
whose structures determine the properties and functions of the
molecules.
 Proteins: The specific order of amino acids in a polypeptide
determines the overall shape of the protein
19

Nucleic acids: Biological information is encoded in sequences of
nucleotide monomers in nucleic acids.
 Each nucleotide is made up of its structural components: ∘ a five-
carbon sugar (Deoxyribose or Ribose) ∘ a Phosphate ∘ a Nitrogen
base (adenine, thymine, guanine, cytosine, or uracil)
 Lipids: Differences in saturation determine the structure and
function of the lipids.
20

 Carbohydrates are biological molecules formed from carbon,
hydrogen, and oxygen, in a ratio of approximately one carbon atom
to one water molecule.
a) Monosaccharide: The most common example is glucose.
Physical properties of monosaccharaides are:
 Monosaccharaides are colorless and crystalline compounds.
 They are readily soluble in water.
 They have a sweet taste.
22

Chemical properties of monosaccharides are:
 Monosaccharides are polyhydroxy aldehydes or Ketones - they are
molecules with more than one hydroxyl group (―OH) and a
carbonyl group (C=O) either at the terminal carbon atom (aldose) or
at the second carbon atom (ketose).
b) Disaccharides: It is produced when two monosaccharides undergo a
dehydration reaction.
Physical properties of disaccharides are:
 Crystalline
 Water-soluble
23

 Sweet to taste
 They are broken into monosaccharides before being absorbed and
used for energy
24

c) Polysaccharides: A long chain of monosaccharides connected by
Glycosidic bonds are called polysaccharides.
The following chemical properties
Not sweet
Many are insoluble in water
Do not form crystals when desiccated
Compact and not osmotically active inside the cells
Can be extracted to create a white powder
25

Lipids
 lipids are made up of carbon, hydrogen, and oxygen, but the atoms are
arranged differently. Most lipids are hydrophobic and nonpolar. Major
lipid types are Fats and Oils; Waxes, Phospholipids, and Steroids.
 Oils and fats, which may be saturated or unsaturated, are healthy and
serve essential plants and animal functions. Lipids don't make polymers
The properties of lipids
 They have minimal or no affinity for water because it consists of
mostly hydrocarbons.
 Highly diverse in function and form. Fats store a large amount of
energy.
26

 Phospholipids are a special type of lipid-linked with cell
membranes and normally have a glycerol backbone
 considered amphipathic because they have both hydrophobic and
hydrophilic components.
 Steroids are classified as lipids based on their hydrophobic
properties.
 Cholesterol is a form of steroid in animal cells’ plasma membrane
27

2.1. Proteins and Their Constituents
 Comes from the Greek word proteios, meaning “first,” or
“primary.”
 Proteins are polypeptides. They are linear chains of amino acids
linked by peptide bonds.
 Some proteins speed up chemical reactions, while
 Others play a role in defense, storage, transport, cellular
communication, movement, or structural support.
28

 Proteins are biopolymers of acids, so named because the amino
group is bonded to α the carbon atom, next to the carbonyl group.
The physical and chemical properties of a protein are determined by
its constituent amino acids.
 An amino acid is an organic molecule with both an amino group
and a carboxyl group
 The individual amino acid subunits are joined by amide linkages
called peptide bonds.
30

 the term is almost always used to refer to an α amino carboxylic
acid. The simplest acid is amino acetic acid, called glycine.
 Other common amino acids have side chains (symbolized by R)
substituted on the carbon atom
 Except for glycine, the acids are all chiral. In all of the chiral
amino acids, the chirality center is the asymmetric carbon atom.
31

 Nearly all the naturally occurring amino acids are found to have the
(S) configuration at the carbon atom.
 They are called L-amino acids because their stereochemistry
resembles that of L-1-2-glyceraldehyde
32

Assigning ‘R and S’ configuration to chiral centers: The Cahn-
Ingold-Prelog rules
 to assign a configuration (R or S) to a chiral carbon enantiomer, let
us be clear of how the spatial arrangement of the tetrahedral carbon,
with respect to the plane of the molecule, is depicted on paper.
33

Step 1: The four groups attached to the chiral carbon atom have to be
arranged in the relative order of priority. The group with the highest
atomic number is given the highest priority #1 and the group with the
lowest atomic number is given the lowest priority i.e. #4. Let’s clarify
this with an example.
34

 Step 2: Make sure that the lowest priority group (#4) is pointing
away from you i.e. it is attached to the hashed wedge in the 2D
spatial arrangement.
 Step 3: Now draw a curved arrow from the highest priority (#1)
substituent to the second lowest priority (#3) substituent.
35

 If the arrow turns in a counterclockwise direction, the configuration
at the stereocenter is labeled S ("Sinister" → Latin= "left"). If,
however, the arrow turns clockwise, then the stereocenter is labeled
R ("Rectus" → Latin= "right").
36

The Standard Amino Acids of Proteins
 The standard amino acids are 20 common -amino acids that are
found in nearly all proteins.
 The standard amino acids differ from each other in the structure of
the side chains bonded to their carbon atoms.
 All the standard amino acids are L-amino acids
37

Essential Amino Acids
 must be provided in the diet
 The ten essential amino acids
 arginine (Arg) valine (Val) methionine (Met) leucine (Leu) threonine
(Thr) phenylalanine (Phe) histidine (His) isoleucine (Ile) lysine (Lys)
tryptophan (Trp)
 Proteins that provide all the essential amino acids in about the right
proportions for human nutrition are called complete proteins.
Examples of complete proteins are those in meat, fish, milk, and
eggs
43

2.2. Nucleic Acids and their Constituents
Nucleic acids
 are the carriers of genetic information
 are the third class of biopolymers
Two major classes of nucleic acids
 deoxyribonucleic acid (DNA): carrier of genetic information
 ribonucleic acid (RNA): an intermediate in the expression of
genetic information and other diverse roles
 RNA comprises of several types namely messenger RNA (mRNA),
ribosomal RNA (rRNA) and transfer RNA (tRNA)
44

RNA play many other roles like
 rRNA is component of ribosomes while
 mRNA contains codons that are translated into proteins.
 tRNA transfers corrects amino acid on the growing polypeptide
chain by reading the codons of mRNA.
The monomeric units for nucleic acids are nucleotides
1. Sugar: ribose in RNA, 2-deoxyribose in DNA
2. Heterocyclic base
3. Phosphate
45

The chemical linkage between monomer units in nucleic acids is a
phosphodiester
46

What are nucleotides?
 the building blocks of nucleic acids:
 they are the monomers which, repeated many times, form the
polymers DNA and RNA
48

Nucleotides = nucleoside + phosphate
A nucleotide comprises a five-carbon sugar molecule:
 deoxyribose in DNA (A) and ribose in RNA (B).
 The carbon atoms on the sugar molecule are numbered in red.
Deoxyribose (A) is different from ribose (B) in that it lacks an –
OH group at carbon 2’.
 The 5’-carbon atom is attached to a phosphate group (here a
monophosphate in orange) and the 1’-carbon is attached to a base
(blue).
49

 A nucleotide may contain more than one phosphate at its 5’-carbon,
for instance the nucleotide adenosine triphosphate has three, as
shown in Figure 2.
 When there is no phosphate group, the molecule is called a
nucleoside.
 Figure 2 | Adenosine triphosphate,
often abbreviated to ATP
50

 Nucleotides of DNA and RNA are made up of nitrogenous bases,
sugar and phosphate.
 The sugar that is present in nucleic acids is pentose sugar which is
of two types; one that present in DNA is 2-deoxy-Dribose and the
other present in RNA is D-ribose.
 Both the pentoses are present as closed five membered ring (β-
furanose form)
51

Structure of pentose sugars present in nucleic acids.
52

 Nitrogenous bases are of two types; purines and pyrimidines.
 A purine is a heterocyclic aromatic organic compound containing 4
nitrogen atoms.
 It contains two carbon rings, and is made of a pyrimidine ring fused
to an imidazole ring.
Structure of a purine
53

 A pyrimidine is a heterocyclic aromatic organic compound
containing 2 nitrogen atoms. It contains only one carbon ring.
Structure of a pyrimidine
 Purines make up two of the four nucleobases in DNA and RNA:
adenine and guanine.
 Pyramidines make up the other bases in DNA and RNA: cytosine,
thymine (in DNA) and uracil (in RNA).
54

 Both purines and pyrimidines have the same function: they serve as
a form of energy for cells, and are essential for production of DNA
and RNA, proteins, starch, regulations of enzymes, cell signaling.
55

 The two purine bases of DNA and RNA are adenine (A) and
guanine (G). Among pyrimidines cytosine (C) is present in both
DNA and RNA, thymine (T) is present in DNA only and uracil (U)
is present in RNA only.
The structure of five major bases is
56

 Nucleotides of both DNA and RNA are covalently linked by
phosphate group in which the 5′-phosphate group of one nucleotide
unit is attached to the 3′ hydroxyl group of the next nucleotide,
forming a phosphodiester linkage
 the alternating phosphate and pentose residues form the covalent
backbones and nitrogenous bases form side groups joined to the
backbone at regular intervals
 The backbone of both DNA and RNA are hydrophilic as –OH
groups of sugar residues form hydrogen bonds with water.
57

Replication of DNA
The Central Dogma (F. Crick): 1957
 Central dogma explains how DNA (genetic instructions)
determined the phenotype.
 According to the central dogma, an index offered by F. Crick, the
genetic instructions are stored as DNA.
 The message in the DNA is converted to RNA during a process
called transcription.
 The message in the RNA is then used to make a protein during a
process called translation.
59

Expression and transfer of genetic information:
 Replication: process by which DNA is copied with very high
fidelity.
 Transcription: process by which the DNA genetic code is read and
transferred to messenger RNA (mRNA). This is an intermediate
step in protein expression
60

 Translation: The process by which the genetic code is converted to
a protein, the end product of gene expression.
 The DNA sequence codes for the mRNA sequence, which codes for
the protein sequence
 mRNA carries information from DNA to the ribosome in a genetic
code that the protein-synthesizing machinery
 translates into protein. Specifically, mRNA sequence is recognized
in a sequential fashion
 Proteins carry out the final actions in the expression, e.g., enzyme
activity, structure of cell or organism, etc.
61

 Genetic information is stored in the molecule deoxyribonucleic
acid, or DNA.
 Four different nucleotide bases occur in DNA: adenine (A),
cytosine (C), guanine (G), and thymine (T).
 The building blocks for DNA synthesis contain three phosphate
groups, two are lost during this process, so the DNA strand contains
one phosphate group per nucleotide.
62

Properties of nucleotides
 strong absorption of UV radiation (260 nm)
 purines are less stable under acidic conditions than pyrimidines
 polar terminal phosphate groups
alternative names:
e.g. adenosine triphosphate (ATP) = adenylate
or adenylic acid
63

2.3. Other Metal-Binding Biomolecules
Hemoglobin and Myoglobin
 The best known iron porphyrin compounds are hemoglobin and myoglobin,
oxygen transfer and storage agents in the blood and muscle tissue,
respectively.
 Myoglobin (Mb) is a globular monomeric protein containing a single
polypeptide chain of 160 amino acid residues (MW 17.8 kDa) made up of
seven α - helical segments (labeled A – G) and six nonhelical segments.
 Hemoglobin (Hb), a tetramer of four globular protein subunits, each of
which is nearly identical to a Mb unit.
 Hb ’ s four subunits are comprised of two α chains of 141 residues and two β
chains of 146 residues with a total molecular weight of 64.5 kDa.
64

 In hemoglobin, α and β chains differ slightly, especially in the manner
in which the porphyrin is held within the protein.
Quaternary structure of deoxyhemoglobin tetramer
 While dioxygen ’ s solubility in water is quite low (6.6 cm3 per liter or
3 × 10 − 4 M), myoglobin and hemoglobin increase O2’ s solubility in
blood approximately 30 times. 66

 Hemoglobin binds dioxygen in a cooperative manner — that is, once
one O2 molecule is bound to the enzyme, the second, third, and fourth
attach themselves more readily.
 Both Mb and Hb bind dioxygen only when the iron ion is in its
reduced state as iron(II).
 The terminology oxy - and deoxy - Mb and Hb refer to the enzyme in
its oxygenated or deoxygenated forms, respectively, both with iron(II)
metal centers, while met - describes oxidized heme proteins containing
iron(III) centers.
67

68
Heme group has iron in the
center in ferrous state , if it is in
ferric state it will not bind to
oxygen
*in ferrous state it can have 6
bonds
4 with nitrogen we call it
porphyrin ring
1 bond with molecular O2
(molecule(O2) NOTE atom(O))
1 attached to the globin chain
(polypeptide

Supply and storage of iron
 In mammals, the task of transferring iron from dietary sources to
haemoglobin initially involves the absorption of Fe(II) after passage
through the stomach, followed by uptake into the blood in the form of
the Fe(III)-containing metalloproteins transferrins.
 Iron is transported as transferrin to protein ‘storage vessels’ until it is
required for incorporation into hemoglobin.
 In mammals, iron is stored mainly in the liver, bone marrow and
spleen in the form of ferritin, a water-soluble metalloprotein.
 Apoferritin has been isolated from, for example, horse spleen and has
a molecular weight of ≈445 000.
70

 X-ray diffraction studies confirm that it consists of 24 equivalent units
(each with 163 amino acid residues) arranged so as to form a hollow
shell, the cavity of which has a diameter of ≈8000pm.
 In ferritin, this cavity contains up to 4500 high-spin Fe3+ centres in the
form of a microcrystalline oxohydroxophosphate of composition
(FeO.OH)8(FeO.H2PO4).
 Results of an EXAFS study indicate that this core comprises double
layers of approximately close packed O2- and [OH]- ions, with
interstitial sites between the layers occupied by Fe(III) centres.
 Adjacent [OFeO]- triple layer blocks are only weakly associated with
each other.
71

 The phosphate groups in the iron-containing core appear to function
as terminators and linking groups to the protein shell.
 While the structures of apoferritin and ferritin are fairly well
established, the manner in which iron is transported in and out of the
protein cavity is still under investigation.
 It is proposed that iron enters as Fe2+ and is oxidized once inside the
protein.
 The formation of the crystalline core is an example of
biomineralization and it is a remarkable achievement of evolution that
iron can be stored in mammals effectively as hydrated iron(III) oxide,
i.e. a form closely related to rust!
72

 The transferrins are glycoproteins (i.e. compounds of proteins and
carbohydrates) and include serum transferrin, lactoferrin (present in
milk) and ovotransferrin (present in egg white).
 In humans, serum transferrin transports 40mg of iron per day to the
bone marrow.
 It contains a single polypeptide chain (molecular weight of ≈80 000)
coiled in such a way as to contain two pockets suitable for binding
Fe3+.
 Each pocket presents hard N- and O-donor atoms to the metal centre,
but the presence of a [CO3]2- or [HCO3]- ligand is also essential.
73

 The stability constant for the Fe3+ complex is very high (log β = 28 at
pH 7.4), making transferrin extremely efficient as an iron transporting
and scavenging agent in the body.
 The exact mechanism by which the Fe3+ enters and leaves the cavity
has not been elucidated, but it seems reasonable that a change in
conformation of the polypeptide chain facilitates the process.
74

Cobalamins (Vitamin and Coenzyme B12)
 For a number of reasons, coenzyme B12 and its derivatives, including
vitamin B12, are useful introductory examples in the field of bioinorganic
chemistry.
 First, several milestones in the development of the field have been
associated with vitamin B12, and later with the coenzyme.
 These pertain to the immediate therapeutic benefit of cobalamins, the early
use of chromatographic purification methods, structural elucidation by x-ray
crystallography and the relationship between enzymatic and coenzymatic
reactivity.
 Furthermore, modern natural product synthesis and both bioorganic and
organometallic chemistry have strongly profited from studies of the B12
system. 75

 It incorporates cobalt into a corrin ring structure, which has one less
=CH bridge between the pyrrole rings than the porphyrins.
76

 Coenzyme B12 is a medium-sized molecule with a molecular mass of
about 1350 Da that exhibits its characteristic specificity and high
reactivity only in combination with corresponding apoenzymes.
 The incorporation of the element cobalt into the coenzyme is quite
surprising, because cobalt is the least abundant first-row (3d)
transition metal in the earth’s crust and in sea water.
 Therefore, a very special functionality is to be expected. 78

 The corrin ligand is also unique, particularly as regards its smaller ring size
compared to the porphin systems.
 Cobalt-containing porphyrin complexes, although stable, are not suitable
for mimicking the actions of coenzyme B12.
 The sixth, axial metal coordination site in coenzyme B12 and
methylcobalamin features a primary alkyl group, which puts these
complexes among the few examples of “natural” organometallic
compounds in biochemistry.
 This compound is known to prevent anemia and also has been found to have
many catalytic properties.
 During the 1920s it was found that injections of extracts from animal liver
were able to cure a very malignant (“pernicious”) form of anemia that could
otherwise be lethal.
79

 Improved methods of trace analysis soon showed that the essential
component of these extracts contained cobalt.
 Since the substance was synthesized only by microorganisms and the
trace element cobalt had to be supplied in any case, the factor was
called “vitamin B12”.
 Enrichment and isolation turned out to be extremely laborious due to
the low concentration of only 0.01 mg vitamin per liter of blood, so
chromatographic separation methods had to be employed.
 During isolation of this compound from natural sources, the adenosine
group is usually replaced by cyanide, and it is in this cyanocobalamin
(vitamin B12) form that it is used medicinally.
80

Enzyme Functions of Cobalamins
 Three different enzyme functions (enzyme classes) are connected with
B12 cofactors, reflecting different types of reactivity.
 The most important enzyme class contains the adenosylcobalamin-
dependent isomerases, which occur in all types of organisms.
 They rely on the homolytical splitting of the Co–C bond of AdoCbl to
form highly reactive radicals.
 The second enzyme class involves methylcobalamin and exhibits
alkylating activity.
81

 The methionine synthase is essential for the biosynthesis of
methionine in many organisms, including humans.
 The third class, B12-dependent reductive dehalogenases (CprA) of
anaerobic microbes, plays an important role in the detoxification of
aromatic and aliphatic organochloro compounds.
 Most of these B12-dependent reductive dehalogenases also contain
Fe/S-clusters, confirming that the B12 cofactor plays the role of a
redox catalyst.
 Since many fundamental questions regarding the reaction mechanism
of dehalogenases still remain unclear, the present discussion will be
restricted to the first two classes of B12-dependent enzymes.
82

Nitrogenase
 The importance of the inorganic-biological nitrogen cycle to life on
earth can hardly be overestimated.
 Only the technical nitrogen fixation realized in ammonia synthesis
(according to F. Haber and C. Bosch) guarantees to supplement the
often growth-limiting nitrogen content of the soil and thus guarantee a
food production adequate for the growing human population.
 Ammonia continues to be one of the leading products of the chemical
industry.
 Other large-scale chemicals such as ammonium nitrate, urea and nitric
acid are follow-up products of the technical “fixation” of nitrogen
obtained from air. 83

 Accordingly, the overall turnover of products from synthetic nitrogen
fixation tends to exceed that of the biological process.
 A further source are physical-atmospherical reactions, which
transform N2 during thunderstorm discharges.
84

(a) Chemical and (b) ecological representation of the nitrogen cycle
85

 Most of the biological systems which participate in the global
nitrogen cycle contain metal-requiring enzymes.
 Three main processes can be distinguished:
 Nitrogen fixation,
 nitrification
 denitrification
86

 The oxidative process of nitrification yields nitrate: the oxidation
state in which nitrogen can be assimilated by most higher plants.
 The crucial challenge in this reaction as performed by Nitrosomas
bacteria is to avoid the formation of the stable dinitrogen molecule,
N2, the zerovalent state.
 Some metalloenzymes for certain stages of denitrification, have
already been introduced: the molybdenum-containing nitrate(N+V)
reductase (→ nitrite), the copper or heme-iron containing nitrite(N+III)
reductases and the copper-containing dinitrogen monoxide(N+I)
reductase.
 Denitrification requires rather anaerobic conditions and organic
substances as reductants. 87

 The final product of denitrification is the extremely stable, inert and
volatile dinitrogen molecule.
 This process, which is comparable only to photosynthesis in its
overall biological importance, is exclusively confined to procaryotic
“diazotrophic” organisms, such as free bacteria of the Azotobacter
strain or Rhizobium bacteria, which exist in symbiosis with the root
system of leguminous plants.
 Due to the thermodynamic stability of the dinitrogen molecule, its
reduction requires a large amount of energy in the form of several ATP
equivalents (with Mg2+ as the hydrolysis catalyst) and (at least) six
electrons per N2 at a physiologically quite negative potential of less
than −0.3V. 88

 In addition, the extremely low reactivity of the dinitrogen molecule requires
efficient catalysts: the dinitrogenase enzymes.
 Due to both thermodynamic and kinetic difficulties, the enzymatic nitrogen
fixation proceeds rather slowly, with turnover numbers on the order of one
per second.
 A large number of stable synthetic complexes of N2 are now known and
Coordination of centrosymmetrical N2 (mostly end-on) requires a two fold
attack:
 in addition to the normal coordinative bond involving the free electron pair
at one nitrogen center and the electrophilic metal ion, the transition metal
center should provide electron density for the relatively low-lying
unoccupied molecular orbitals of the triple-bond system in N≡N via π
interactions. 89

 Thus, N2 complexation and fixation requires d electron-rich metal
centers, as found for example in many organometallic compounds.
 The low redox potentials and the necessarily high reactivity of
nitrogenase enzymes further require the absence of competing
molecules such as dioxygen, O2. 90

 Therefore, N2-assimilating microorganisms are either obligatory anaerobes
or posses complex protection mechanisms for the exclusion of O2 from the
active site of nitrogenase enzymes.
 These mechanisms include iron-containing proteins, which serve as O2
sensors.
 The nitrogenase enzymes responsible for nitrogen fixation contain two
proteins.
 The iron-molybdenum protein contains two metal centers.
 One, called the FeMo-cofactor, contains molybdenum, iron, and sulfur.
 This may be the site of nitrogen reduction; there are open binding sites on
some of the iron atoms in the middle and a pocket large enough for substrate
binding. 91

 The other site, called the P-cluster, contains eight iron atoms and
eight sulfur atoms in two subunits that are nearly cubic.
 The P-cluster is believed to assist the reaction by transfer of electrons,
but little more is known about the mechanism of the reaction.
 The second protein contains two identical subunits with a single
4Fe:4S cluster and an adenosine diphosphate (ADP) molecule bound
between the two subunits.
 In some fashion, this protein is reduced and transfers single electrons
to the FeMo-cofactor protein, where the reaction with nitrogen takes
place.
 Eight electrons are required for N2 conversion to 2NH3 by the
enzymes because the reaction also forms H2. 92

 The nitrogenase reaction seems to begin with a series of four electron
transfers, leading to a reduced form of the enzyme that then can bind
four H+ ions.
 After these changes, N2 can be bound as H2 is released and, finally,
the N2 is reduced to NH3 and released from the complex.
 Two alternative sites for the first proton to bind have been suggested,
one in the middle of the Fe & cluster in the MoFe active site, and the
other at an alkoxy oxygen of homocitrate bound to Mo at one end of
the cluster.
93

 Calculations show the possibility of N2 bonding asymmetrically with one of
the N atoms near the center of the four Fe atoms, approximately at the
corners of a square on the front face and the other sticking out toward the
top front.
 This more distant N is positioned to accept H atoms from the nearby S
atoms, which could release NH3.
 The remaining N can then accept H atoms in a similar fashion from S atoms
to form the second NH3.
 There have been many attempts to make model compounds for ammonia
production, but none have been successful.
 How the enzyme manages to carry out the reaction at ambient temperature
and less than 1 atm pressure of N2 is still an unanswered question.
94

Photosynthesis
 Photosynthesis is fundamental for the existence of higher forms of life
on earth; the production of reduced carbon compounds on the one
hand and the production of oxygen on the other are based on this
radiation energy-consuming process.
 Certain bacteria and algae are photosynthetically active, as are green
plants. 95

 These bacteria use photosynthesis primarily to separate charges
and thus create a transmembrane proton gradient (pH difference),
which is used to synthesize high-energy adenosine triphosphate
(ATP) from adenosine diphosphate (ADP phosphorylation
96

 Chlorophylls contain Mg2+ as the central ion and partially
hydrogenated and substituted porphyrin ligands (“chlorins”) with an
annelated five-membered ring.
 Chlorophyll molecules differ slightly chlorin ring is with one double
bond reduced porphine ring with regard to some substituents.
97

Primary Processes in Photosynthesis
Light Absorption (Energy Acquisition)
 An efficient photosynthetic transformation of light energy(1.24–3.26
eV) requires the absorption of as many photons as possible.
 This condition is fulfilled through the presence of various organic
pigments, including chlorophyll molecules, which are positioned in a
highly folded membrane with an inherently large inner surface area
and therefore a high cross-section for photon capture.
 Chlorophylls contain a conjugated tetrapyrrole system, which shows
a high absorptivity at both the long- and short-wavelength ends of the
visible spectrum.
99

 Bacteriochlorophylls, which contain two partially hydrogenated
pyrrole rings in contrast to the “normal” chlorophylls with only one
dihydropyrrole ring, absorb light at particularly lowenergy in the near
infrared region, an effect which can also be achieved by formyl
substituents.
Absorption spectra of some pigments from algae and plants 100

Exciton Transport (Directed Energy Transfer)
 The absorption of energy in the form of photons by pigments requires less
than 10−15 seconds and yields short-lived electronically excited (singlet)
states that, in principle, can produce a charge separation.
 The major part (>98%) of the chlorophyll molecules should act as “antenna”
devices and collect available photons (“light harvesting”)
 There must be efficient and spatially oriented (“vectorial”) transfer of the
absorbed energy in the form of excited states (“excitons”) to the actual
reaction centers, which contain less than 2% of the total chlorophyll content.
 The energy transfer, which requires neither mass nor charge movement, is
made possible by a special arrangement of many chlorophyll chromophors
in a network of “antenna pigments”.
101

 These chromophors are arranged in spatial proximity and a certain,
well-defined orientation; they are able to “tunnel” the light energy to
the actual reaction centers with about 95% efficiency within 10–100
ps.
Light-harvesting architectures for Rhodopseudomonas acidophila 102

 The role of magnesium in chlorophyll is to contribute to the
particular arrangement of pigments.
 The virtually loss-free exciton transfer through a cluster network of
antenna pigments requires a high degree of three-dimensional order.
 Therefore, a well defined spatial orientation of the chlorophyll
chromophors with regard to each other is necessary.
 This orientation cannot be solely guaranteed by anchoring the
chlorophyll molecules in the membrane via the phytyl side chains but
must rely also on the coordination of polypeptide side-chain ligands to
the two free axial coordination sites at the metal (three point fixing for
defined spatial orientation).
103

 The coordinatively doubly unsaturated Lewis acidic Mg2+ centers can
interact via dipolar, hydrogen-bonding water molecules with the
Lewis basic carbonyl groups in the characteristic cyclopentanone ring
of adjacent chlorophyll molecules.
 Exactly defined spatial orientation, such as the arrangement of
tetrapyrrole ring planes parallel to the plane of the membrane and the
resulting high degree of organization in light-harvesting systems, is
thus ensured by the presence of a coordinatively doubly unsaturated
electrophilic metal center in the macrocycle.
 Of all the metals with the proper size, sufficient natural abundance and
strong tendency for six-coordination, only Mg2+ remains of all the
main-group metal ions.
104

 In addition, magnesium is a rather light atom, with a small spin–orbit
coupling constant.
 Heavier elements, including main-group metal ions, have higher spin–
orbit coupling constants and would thus enhance an intersystem
crossing (ISC) from very short-lived singlet to considerably longer-
lived triplet excited states, thereby slowing down the necessarily very
rapid primary events in photosynthesis.
 The result would be a competition of undesired light- or heat-
producing processes with the actual chemical reactions.
 The fact that transition metals in particular are not suited to being the
central ions of chlorophylls is related to the next step of
photosynthesis: the charge separation.
105

Charge Separation and Electron Transport
 When excitonic energy reaches a photosynthetic “reaction center”, the
essential step for the separate production of an electron-rich
component and an electron-poor species can take place.
 Purple bacteria, such as Rhodopseudomonas viridis, which contain
only one photosystem have their reaction center situated in a
polyprotein complex that in vivo spans the membrane.
 On the symmetry axis of the nearly C2-symmetrical reaction centers is
a BC dimer, the “special pair” BC/BC.
 Structure determination indicates that the aggregation can result from
a coordinative interaction between acetyl substituents at the
polypyrrole ring and the metal centers.
106

 Because of the significant – orbital interaction, the special pair may
function as an electron donor; electronic excitation of this pair, which
is also called P960 (pigment with a long-wavelength absorption
maximum at 960 nm),
 leads to a primary charge separation within a very short time: one
energetically elevated electron of the electronically excited dimer is
transferred to the primary acceptor, a monomeric BC molecule.
 The next step of the charge separation consists the transfer of negative
charge to the secondary acceptor bacteriopheophytin (BP), a BC
ligand without coordinated metal.
107

 From the coordination chemistry of porphyrins, it is well known that
neutral M2+ complexes are often harder to reduce than corresponding
doubly protonated neutral ligands; the more ionic bond to the metal
leaves considerable amounts of negative charge at the ligand.
 Since the central ion Mg2+ is redox-inert and thus not directly
involved in electron donation or acceptance, the radical anions of
chlorophylls can be regarded as complexes of a divalent metal cation
and the radical trianion of the macrocyclic ligand: (Chl/Mg)•−
=Chl•3−/Mg2+.
 Correspondingly, radical cations of tetrapyrrole complexes can be
formulated as compounds of metal dications with anion radical
ligands.
108

 The third detectable acceptor for the electron generated through light-
induced charge separation in the reaction center is a para-quinone, Qa,
such as menaquinone, which is reduced to a semiquinone radical
anion, Qa•−, in this process.
 Reduced Qa may in turn reduce a different, more labile quinone, such
as ubiquinone, Qb, with a high-spin iron(II) center connecting the two
quinones at the axis of the reaction center.
 Possibly, a divalent metal ion is required to guarantee the controlled
electron transfer from reduced BP to the primary quinone through
polarization of the hydrogen bond-forming histidine ligands or simply
to maintain the necessary structure.
109

 Quinone Qb, or its 2e−/2H+ reaction product, the hydroquinone H2Qb, is not
tightly bound to the protein but exchanges with quinones in the “quinone
pool” of the membrane, so that electrons can now be transported further
outside the protein.
 In simple bacteria, the electron gradient finally gives rise to a coupled H+
gradient and photosynthetic phosphorylation takes place.
 In higher organisms there are further steps, the “dark reactions”, which
eventually lead to the production of the electron-rich coenzyme NADPH and
to Mg2+-requiring CO2 reduction (Calvin cycle).
 The formation of long-lived, storable oxidation and reduction products as a
consequence of light-induced charge separation is very unusual, the normal
course of events being a rapid recombination of charges to produce heat or
light (emission, luminescence). 110

 The success of photosynthesis is based on the strong preference for the
extremely rapid charge-separating steps rather than the slower, energy-
releasing recombination processes.
 From the absolute necessity of preventing heat- or light-producing back
electron-transfer reactions, it follows that the chlorophyll molecules must
not contain a redox-active transition metal.
 Metal centers like FeII/III, which easily transfer electrons themselves, could
accept or donate electrons in the ground or electronically excited states of an
Fe-chlorophyll system and thus preclude the photosynthesis, which requires
a rapid spatial charge separation.
 An intramolecular instead of an intermolecular electron transfer would
result, without chemical energy storage. 111

 The role of the magnesium ion in chlorophylls is therefore to act as a
light, redox-inert, Lewis acidic coordination center that contributes to
a defined three-dimensional organization in light-harvesting systems
and reaction centers.
 Monomers, π–π dimers and metal-free ligands each have separate,
unique functions in the primary processes of photosynthesis.
 Redox-active transition metals or heavier metal centers with higher
spin–orbit coupling constants would support additional, undesired
reaction alternatives; of the remaining bioavailable metal ions, only
Mg2+ fits exactly with regard to size and charge.
112

3. Principles of Coordination in Bioinorganic Systems
Reading Assignment
113

4. Characterization of Biological Molecules
 Can be cell-based and noncell based and analytical techniques
 to obtain a fundamentally deeper and broader understanding of
biological relationships and approaches for structural analysis and
design of biological molecules
Noncell based analytical techniques
Surface plasmon resonance
 for analyzing the binding properties of the biomolecules
 It is an excellent instrumentation for a label-free, real-time
investigation of molecular interactions
114

 for the analysis of the functional properties of biomolecule
 technology in affinity biosensors for biomolecular interaction
analysis (BIA)
 BIA with biosensors allowed a real-time follow-up of the kinetics
of interaction between a pair of biomolecules in absence of any
tracer
 This was made possible by immobilizing one of the partner of the
pair on a metal surface, allowing the other partner to flow in excess
over that surface, whilst using SPR spectroscopy to measure the
changes in refractive index occurring at the metal surface upon
interaction between the two biospecific partner 115

 The SPR-based binding method involves immobilization of a ligand
on the surface of a sensor chip using well-defined chemistry
(direct chemical coupling or capture-based method) and allowing
solutions with different concentrations of an analyte to flow over
it and to characterize its interactions to the immobilized ligand
Operating Principle of SPR Biosensors
 Surface plasmon resonance occurs when a photon of incident light
hits a metal surface (typically a gold surface)
 At a certain angle of incidence, a portion of the light energy
couples through the metal coating with the electrons in the metal
surface layer, which then move due to excitation
116

 The electron movements are now called plasmon, and they
propagate parallel to the metal surface. The plasmon oscillation in
turn generates an electric field whose range is around 300 nm from
the boundary between the metal surface and sample solution
 The defined SPR angle, at which resonance occurs, on the
conditions of the constant light source wavelength and metal thin
surface, is dependent on the refractive index of the material near the
metal surface
117

 Consequently, when there is a small change in the reflective index
of the sensing medium (e.g., through biomolecule attachment),
plasmon cannot be formed.
 Detection is thus accomplished by measuring the changes in the
reflected light obtained on a detector.
 In addition, the amount of surface concentration can be quantified
by monitoring the reflected light intensity or tracking the resonance
angle shifts
118

Figure 1. Concept of a surface plasmon resonance (SPR) biosensor: (A)
Kretschmann geometry of the ATR method; (B) spectrum of reflected light before
and after refractive index change; (C) analyte-biorecognition elements binding on
SPR sensor surface and (D) refractive index changes caused by the molecular
interactions in the reaction medium.
119

 The technique presents several advantages over conventional
methods of affinity measurements.
 It requires little material, is very fast and does not need any labeling
with a tracer.
 A major drawback is the need to regenerate the sensor chip for
measurements with various concentrations
120

Electrophoresis
 Electrophoresis is a simple, sensitive and rapid analytical tool
commonly used to separate the charged biomolecules such as DNA,
RNA or proteins according to their size and charge properties.
 The technique works on the principle that charged molecules under
the influence of electric field migrate towards oppositely charged
electrodes.
 The positively charged molecules move toward cathode and
negatively charged molecules move toward anode.
 The migration is due to the charge on the molecules, size of the
molecules and potential applied across the electrodes 121

The electrophoresis can be broadly divided into two categories
Gel electrophoresis
 uses electricity to separate DNA fragments by size as they migrate
through a gel matrix. Polyacrylamide and agarose are two support
matrices commonly utilized in electrophoresis.
 The gel matrices serve as porous media and play a role as a
molecular sieve to separate different sizes of proteins and nucleic
acids.
 Agarose has a large pore size and good range of separation and is
suitable for separating nucleic acids and protein complexes
122

 Polyacrylamide has a smaller pore size, higher resolving power and
is typically recommended for separating proteins and smaller
nucleic acids.
 Polyacrylamide gel electrophoresis (PAGE) is usually carried out
in the presence of the denaturing agent referred as sodium dodecyl
sulfate (SDS)
 the samples are loaded into wells at one end of a gel, and under the
influence of the applied electric field, the samples are pulled
through the gel
 Depending on the charge on the biomolecule, it moves toward the
opposite electrode.
123

 In the case of same amount of charge per mass as in SDS-PAGE,
small fragments move through the gel faster than large ones on the
basis of their molecular mass. Upon staining gel with a DNA or
protein binding dye, the biomolecular fragments can be seen as
bands.
Capillary electrophoresis
The capillary electrophoresis (CE) is a high resolution electrophoresis
technique that uses narrow-tube (20–200 μm i.d.) capillaries to
perform separations of biomolecules under the influence of electric
field.
124

 The molecules are separated based on their electrophoretic mobility (e/m
ratio).
 The electrophoretic mobility is dependent upon the charge of the
molecule, pH and viscosity of the separation medium, and the
hydrodynamic radius of the molecule
 The rate at which the particle moves is directly proportional to the applied
electric field-the greater the field strength, the faster the mobility
 Compared with gel electrophoresis, stationary support is not required in
CE, number of samples and reagents consumed in CE is minute, better
power of separation and samples can be immediately analyzed by the
detectors positioned at the end of the capillary tubes
125

Cell-based analytical techniques
Flow cytometry
 FC is powerful laser-based technology that measures cells as they
flow in a liquid stream through a beam of light.
 Cells are fluorescently labeled and then excited by the laser to emit
light at varying wavelengths.
 The fluorescence of an individual cell can be measured and
analyzed, providing output measurements for each cell, rather than
for the whole sample population
126

 FC is a fast and reproducible technique that can analyze up to
thousands of cell particles per second as they pass through the
liquid stream
Fluorescence activated cell sorting
 is a specialized type of FC method that sort heterogeneous mixture
of cells into different containers based upon the scattering and
fluorescent characteristics of each cell.
 The cell suspension flows through a narrow stream of liquid.
127

 The individual cell passes through a point where the fluorescent
character of each of the cell is measured which is followed by
application of a vibrating mechanism resulting in stream of cells
separating into individual cells.
 An electric charge ring positioned right below the vibrating
mechanism produce a charge based on the prior fluorescent
measurements and an opposite charge is given on the droplet as it
breaks from the stream.
 The charged droplets are directed and distributed into different
containers based on their charge via electrostatic deflection system
128

Analytical techniques to identify & characterize biological
molecules
Mass spectrometry
 not only identification but also in quantification of proteins
 There are two approaches to mass spectrometric analysis of a
proteome for protein identification
 The bottom up approach includes separation of proteins of a
mixture followed by enzymatic / chemical generation of their
peptides, and evaluation of separated peptides on a mass
spectrometer
129

 In the top down proteomics, intact proteins are introduced into the
mass spectrometer and proteins are fragmented directly inside the
mass spectrometer
 MS is based on the ability of the mass spectrophotometer to assess
the mass-to-charge ratio (m/z) of ions.
 The mass spectrophotometer consists of an ion source (ionizes the
peptides), a mass analyzer (separates the ions on the basis of m/z
ratio) and a detector (registers the data)
130

NMR spectroscopy
 NMR spectroscopy is a powerful tool to elucidate the structure,
interactions and dynamics of macromolecules
 Elements that possess magnetic moment, due to the spin of
positively charged nucleus, can be detected by NMR spectroscopy
by keeping either the magnetic field strength or radio frequency
constant and sweeping the other over a range of values
 A small chemical shift in the spectrum arises as variations in the
magnetic field occur due to interaction of orbiting electrons with
the nucleus in varying chemical environment
131

 On analysis, the chemical shift helps in interpreting the functional
group as well as strength of its interaction in the molecule as
predicted by the available data on the range of chemical shift
values.
 NMR spectrometer basically comprises of a cryomagnet, a
transmitter and a detector.
 The field strength of the magnet is related to the sensitivity and
resolution of NMR spectra.
 The transmitter provides radio frequency pulses to irradiate the
samples with a desired pulse length (or width).
132

 A receiver detects the NMR signal generated at the probe and
amplifies the signal so that it becomes amenable for digitization.
 Its advantages is
 The technique is a noninvasive, nondestructive, highly reproducible
and does not require complex sample preparation methods,but
 Its disadvantage is
 It has low sensitivity
133

X-ray crystallography
 is a technique that enables the determination of 3D structure of
molecules. Also, the experimental determination of 3D structure of
drug targets has helped gain knowledge in the field of drug
discovery.
 Data are collected on the basis of diffraction pattern obtained from
the scattering of X rays, due to electron density of the molecule,
when passed through ordered and regular arrangement of
atoms/molecules in a crystal
134

 Data are collected on the basis of diffraction pattern obtained from
the scattering of X rays, due to electron density of the molecule,
when passed through ordered and regular arrangement of
atoms/molecules in a crystal.
 The pattern of the diffraction spots provides information about the
crystal packing symmetry and repeating unit of the crystal
135

6. Metalloenzymes containing Fe, Zn, Ni, Co, Cu and Mo, role of
these enzymes in biology, their structural and mechanistic aspects
 Metalloproteins and Metalloenzymes
 A protein that contains one or more metal ions tightly bound to
amino acid side chains is called a metalloprotein;
 some of the most common ligands provided by amino acids are
shown here.
 A metalloprotein that catalyzes a chemical reaction is a
metalloenzyme.
137

6.1. Electron-Transfer Proteins
 Proteins whose function is to transfer electrons from one place to
another are called electron-transfer proteins.
 Because they do not catalyze a chemical reaction, electron-transfer
proteins are not enzymes; they are biochemical reductants or
oxidants consumed in an enzymatic reaction.
 The general reaction for an electron-transfer protein is as follows:
138

 Because many transition metals can exist in more than one
oxidation state, electron-transfer proteins usually contain one or
more metal ions that can undergo a redox reaction.
 Incorporating a metal ion into a protein has three important
biological consequences
 The protein environment can adjust the redox potential (E0′), of the
metal ion over a rather large potential range, whereas the redox
potential of the simple hydrated metal ion [Mn+(aq)], is essentially
fixed.
 The protein can adjust the structure of the metal complex to ensure
that electron transfer is rapid
139

 The protein environment provides specificity, ensuring that the
electron is transferred to only the desired site
 Three important classes of metalloproteins transfer electrons: blue
copper proteins, cytochromes, and iron–sulfur proteins, which
generally transfer electrons at high (> 0.20 V), intermediate (±0 V),
and low (−0.20 to −0.50 V) potentials, respectively
 Although these electron-transfer proteins contain different metals
with different structures, they are all designed to ensure rapid
electron transfer to and from the metal.
 hus when the protein collides with its physiological oxidant or
reductant, electron transfer can occur before the two proteins
diffuse apart 140

Electron Carriers
Iron-Sulfur Proteins
 the major function of the iron-sulfur cluster is to facilitate electron transfer
 These enzymes usually, but not always, have additional metal centers at
which atom-transfer reactions take place.
 The best characterized example of an enzyme that has only an iron-sulfur
center at its active site is aconitase
 the reaction catalyzed by this enzyme
 does not appear to involve electron-transfer processes at any stage; yet a
unit nearly identical to one found in pure redox iron-sulfur proteins is
utilized
141

 The oxidation-reduction chemistry of iron-sulfur proteins and the
corresponding synthetic models for their nFe-mS cores have been
studied extensively.
 The four known classes of iron-sulfur proteins will be discussed in
order of increasing number of iron atoms in the cluster.
(i) [IFe-OS] the rubredoxins have active sites that consist of a high-
spin Fe(I1,III)ion coordinated to four cysteinate sulfur atoms in
an approximately tetrahedral array.
(ii) (ii) [2Fe-2S]. The crystal structure of S. plafensis ferredoxin
revealed the presence of an {Fe,S,(S-cys)4,} unit
142

 The oxidized form of this site contains two Fe(II1)ions, each of
which is tetrahedrally coordinated by two bridging sulfido Froups
and by two terminal cysteine thiolates.
 The iron-iron distance is 2.70 A with Fe-S-Fe angles near 75",
reflecting the presence of significant metal-metal bonding.
(iii) [3Fe-4S]. The events leading to the characterization of three-iron
centers in iron-sulfur proteins.
Well-refined crystal structures are now available for several proteins
that contain the Fe,S,(Scys)3, unit.
143

(iv) [4Fe-4S]. The final type of well-characterized iron-sulfur center
has the Fe,S, cubane-type structure. These clusters can be visualized
as a dimer of two Fe2S2 units, of the kind found in plant-type
ferredoxins, joined so that the sulfido groups now bridge three rather
than two iron centers.
144

5. Metal Ion Folding and Cross-Linking of Biomolecules
 Metals can act as templates for organizing three-dimensional
biological structures. In binding to protein sites, metal ions discard
most, if not all, of their coordinated water molecule.
 When interacting with nucleotides and nucleic acids, many of the
water ligands remain and help organize the structure.
 The tertiary structures induced by metal binding can facilitate
interactions between macromolecules
145

 The formation of cross-links is important for the mechanism of
action of some inorganic-element-based drugs.
 The large ionic radii of metal ions (versus the proton) and the
ability to afford high coordination numbers are used to advantage.
146

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