BiologicalSymbiotic Non Symbiotic
AbiologicalIndustrialNatural
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Abiological nitrogen fixation In abiological nitrogen fixation
the nitrogen is reduced to ammonia without involving any living
cell. Abiological fixation can be of two types : industrial and
natural. For example, in the Habers process, synthetic ammonia is
produced by passing a mixture of nitrogen and hydrogen through a
bed of catalyst (iron oxides) at a very high temperature and
pressure. In natural process nitrogen can be fixed especially
during electrical discharges in the atmosphere. It may occur during
lightning storms and nitrogen in the atmosphere can combine with
oxygen to form oxides of nitrogen.
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Biological Nitrogen Fixation Symbiotic Root nodule Formation
Enzyme ActionNon Symbiotic Biological nitrogen fixation Conversion
of Atmospheric nitrogen (N=N) is reduced to ammonia in the presence
of nitrogenase.
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Nitrogenase is a biological catalyst found in some
microorganisms. such as the symbiotic Rhizobium and Frankia, or the
free-living Azospirillum and Azotobacter. Biological nitrogen
fixation is brought about both by free-living soil microorganisms
and by symbiotic associations of microorganisms with higher plants.
Leguminous plants fix atmospheric nitrogen by working symbiotically
with special bacteria, rhizobia, which live in the root nodules.
Rhizobia infect root hairs of the leguminous plants and produce the
nodules. The nodules become the home for bacteria where they obtain
energy from the host plant and take free nitrogen from the soil air
and process it into combined nitrogen. In return, the plant
receives the fixed N from nodules and produces food and forage
protein.
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Mechanism of biological nitrogen fixation Nitrogen fixation
requires: i.The molecular nitrogen ii.A strong reducing power to
reduce nitrogen like FAD (Flavin adenine Dinucleotide iii.Source of
energy (ATP) to transfer hydrogen atoms to dinitrogen and
iv.Enzymes nitrogenase and Legheamoglobin v.Compound for trapping
the ammonia formed since it is toxic to cells. vi.The reducing
agent and ATP are provided by photosynthesis and respiration.
Proteins and their Relation to life DNARNAPROTEIN
STRUCTUREANTIBODIES STORAGE TRANSPORT ENZYMES
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Myoglobin, Hemoglobin, Cytochromes bind O2 Oxygen is
transported from lungs to various tissues via blood in association
with hemoglobin In muscle, hemoglobin gives up O2 to myoglobin
which has a higher affinity for O2 than hemoglobin. Oxygen-binding
curve for hemoglobin is sigmoidal whereas for myoglobin it is
hyperbolic. This facilitates transfer of O2 to myoglobin.
Cytochromes participate is redox reactions and are components of
the electron transport chain. Oxygen-Binding Proteins
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Hemoglobin is a O 2 transport protein found in the RBCs An
oligomeric protein made up of 2 dimers, a total of 4 polypeptide
chains: 1122. Molecular weight : 64,500. The (141 aa) and (146 aa)
subunits have < 50 % identity. The 3D- structures of (141 aa)
and (146 aa) subunits of hemoglobin and the single polypeptide of
myoglobin are very similar; all three are members of the globin
family. Each Hb subunit consists of 7 () or 8 () alpha helices and
several bends and loops folded into a single globin domain. Each
subunit has a heme-binding pocket. Hemoglobin Structure
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The heme group is responsible for the O 2 -binding capacity of
hemoglobin. The heme group consists of the planar aromatic
protoporphyrin made up of four pyrrole rings linked by methane
bridges. A Fe atom in its ferrous state (Fe +2 ) is at the center
of protoporphyrin. Fe +2 has 6 coordination bonds, four bonded to
the 4 pyrrole N atoms. The nucleophilic N prevent oxidation of Fe
+2. The two additional binding sites are one on either side of the
heme plane. One of these is occupied by the imidazole group of His.
The second site can be reversibly occupied by O 2, which is
hydrogen bonded to another His. The Prosthetic Heme Group
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When hemoglobin is bound to O 2, it is called oxyhemoglobin.
This is the relaxed (R ) state. The form with a vacant O 2 binding
site is called deoxyhemoglobin and corresponds to the tense (T)
state. If iron is in the oxidized state as Fe +3, it is unable to
bind O 2 and this form is called as methemoglobin CO and NO have
higher affinity for heme Fe +2 than O 2 and can displace O 2 from
Hb, accounting for their toxicity. Different forms of
Hemoglobin
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Hemoglobin exists in two major conformational states: Relaxed
(R ) and Tense (T) R state has a higher affinity for O2. In the
absence of O2, T state is more stable When O2 binds, R state is
more stable, so hemoglobin undergoes a conformational change to the
R state. The structural change involves readjustment of
interactions between subunits. T and R states of Hemoglobin
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O2 binding rearranges electrons within Fe+2 making it more
compact so that it fits snugly within the plane of porphyrin. Since
Fe is bound to histidine of the globin domain, when Fe moves, the
entire subunit undergoes a conformational change. This causes
hemoglobin to transition from the tense (T) state to the relaxed
(R) state. The 11 and 22 dimers rearrange and rotate approximately
15 degrees with respect to each other Inter-subunit interactions
influence O2 binding to all 4 subunits resulting in cooperativity.
Changes Induced by O2 Binding
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Four subunits, so four O2-binding sites O2 binding is
cooperative meaning that each subsequent O2 binds with a higher
affinity than the previous one Similarly, when one O2 is
dissociated, the other three will dissociate at a sequentially
faster rate. Due to positive cooperativity, a single molecule is
very rarely partially oxygenated. There is always a combination of
oxygenated and deoxygenated hemoglobin molecules. The percentage of
hemoglobin molecules that remain oxygenated is represented by its
oxygen saturation. O2-binding curves show hemoglobin saturation as
a function of the partial pressure for O2. O2-binding kinetics
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Myoglobin OXYGEN TRANSPORT
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A simple oxygen-binding protein found in almost all mammals,
primarily in muscle tissue. Myoglobin (Mr 16,700; abbreviated Mb)
As a transport protein, it facilitates oxygen diffusion in muscle.
Myoglobin is abundant in the muscles of diving mammals such as
seals and whales Also has oxygen storage function for prolonged
excursions undersea. A single polypeptide of 153 amino acid
residues with one molecule of heme. It is typical of the family of
proteins called globins, all of which have similar primary and
tertiary structures. The polypeptide is made up of eight -helical
segments(78%) connected by bends. Myoglobin - Structure
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Chlorophyll PORPHYRINES
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Porphyrins (pronounce) are tetrapyrroles. They consist of four
weakly aromatic pyrrole (pronounce) rings joined by methene
bridges. Porphyrin is a heterocyclic macrocycle made from 4 pyrrole
subunits linked on opposite sides ( position) through 4
methinebridges (=CH-). The extensive conjugated system makes the
compound chromatic, hence the name porphyrin, from a Greek word for
purple The macrocycle has 22 pi electrons, 18 of which are active
in the conjugated system. These are the central groups of
biologically imp molecules such as Hemoglobin, Myoglobin,
Chlorophyll, Cytochromes, etc Porphyrins
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Because of the unique chemistry of porphyrins, they are able to
perform Several functions: As a metal binder (ligands) As a solar
cell (convert light or chemical energy) As an oxygen transport
medium (hemoglobin) As an electron transfer medium (conducting
polymers) Gene regulation Drug metabolism Iron metabolism Hormone
synthesis Uses of Porphyrins
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As the basic building block of hemoglobin Heme a cross-coupled
porphyrin used in the larger molecule hemoglobin
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Cytochrome C a molecule responsible for transporting an
electron used to provide energy to the organism. These molecules
are identical, or very similar, for related species of plants or
animals.
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A green compound found in leaves and green stems of plants. It
occurs in cell organelles called chloroplasts, which are absent in
animals. Chlorophyll is the molecule that traps this 'most elusive
of all powers and is called a photoreceptor. The basic structure of
a chlorophyll molecule is a porphyrin ring, coordinated to a
central atom. This is very similar in structure to the heme group
found in hemoglobin, except that in heme the central atom is iron,
whereas in chlorophyll it is magnesium. Chlorophyll
Slide 39
Chlorophyll a and chlrophyll b The most important pigment in
plants is chlorophyll. Two types of chlorophyll in plants,
chlorophyll a (chl a) and chlorophyll b (chl b) Chlorophyll is
composed of two parts; the first is a porphyrin ring with magnesium
at its center, the second is a hydrophobic phytol tail The ring has
many delocalized electrons that are shared between several of the
C, N, and H atoms; these delocalized electrons are very important
for the function of chlorophyll. The tail is a 20 carbon chain that
is highly hydrophic and stabilizes the molecule in the hydrophobic
core of the thylakoid membrane.
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Structurally CH 3 group is present in chl a where chl b has a
CHO group. Chlorophyll a and b absorb different wavelengths better
than others. chl a absorbs best at 450 and 680 nm chl b absorbs
best at 500 and 640 nm chlorophyll a is directly involved in the
redox reactions of the light reactions, chl b functions as an
accessory pigment Accessory pigments absorb light and pass the
energy from the light to the chl a in the reaction center Other
accessory pigments can be present such as xanthophylls and the more
well known carotenoids. The most well known carotenoid is
beta-carotene which absorbs different wavelengths than the
chlorophylls.
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Photosystems Within the thylakoid membranes of the chloroplast,
are two photosystems. Photosystem I optimally absorbs photons of a
wavelength of 700 nm. Photosystem II optimally absorbs photons of a
wavelength of 680 nm. Photosystem II uses light energy to oxidize
two molecules of water into one molecule of molecular oxygen. The 4
electrons removed from the water molecules are transferred by an
electron transport chain to ultimately reduce 2NADP+ to 2NADPH.
During the electron transport process a proton gradient is
generated across the thylakoid membrane. This proton motive force
is then used to drive the synthesis of ATP. This process requires
PSI, PSII, cytochrome bf, ferredoxin-NADP+ reductase and
chloroplast ATP synthase.
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Chlorophyll A and B absorb light mostly in the red and blue
regions of the spectrum Carotene and xanthophyll absorb light from
other regions and pass the energy to chlorophyll
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Metalloenzymes
Slide 49
Introduction Metals play roles in approximately one-third of
the known enzymes. Metals may be a co-factor or they may be
incorporated into the molecule, and these are known as
metalloenzymes. Amino Acids in peptide linkage posses groups that
can form coordinate-covalent bonds with the metal atom. The free
amino and carboxy group bind to the metal affecting the enzymes
structure resulting in its active conformation (2). Metals main
function is to serve in electron transfer. Many enzymes can serve
as electrophiles and some can serve as nucleophilic groups. This
versatility explains metals frequent occurrence in enzymes. Some
metalloenzymes include hemoglobins, cytochromes,
phosphotransferases, alcohol dehydrogenase, arginase, ferredoxin,
and cytochrome oxidase. Carboxypeptidase A is a zinc metalloenzyme
that breaks peptide linkages in the digestion of proteins. The zinc
ion that the enzyme contains in its active site plays a key role in
that function. Metalloenzymes can be regulated in several ways
since they are such a diverse group. One way metalloenzymes are
regulated is the pH level. The pH level can disrupt the electron
flow that the metal would normally help facilitate. In this way the
pH level could inhibit the overall effectiveness of the
metalloenzyme. Transition state analogs play a key role in the
competitive inhibition of metalloenzymes because they mimic the
structure of the substrates transition state in the reaction of
enzyme and substrate. Metalloenzymes such as the ones containing
zinc can also be regulated by diet. The source of zinc in humans is
almost entirely through diet. Without proper intake of metals such
as zinc in a persons diet, the activity of the enzyme would be
inhibited. One thing to keep in mind while studying metalloenzymes
is that they are incredibly diverse and function in a multitude of
important physiological processes
Slide 50
Structure and Overview Metalloenzymes are proteins which
function as an enzyme and contain metals that are tightly bound and
always isolated with the protein. In proteins such as hemoglobins
and cytochromes, the metal is Fe2+ or Fe3+, and it is part of the
heme prosthetic group. In other metalloenzymes the metal is built
into the structure of the enzyme molecule. The metal ion can not be
removed with out destroying the structure of the enzyme. Metals
built into the molecule include: most phosphotransferases,
containing Mg2+; alcohol dehydrogenase, Zn2+; arginase, Mn2+;
ferredoxin, Fe2+; and cytochrome oxidase, Cu2+ (9). Metals are
usually found in the active site of the enzyme. The metals resemble
protons (H+) in that they are electrophiles that are able to accept
an electron pair to form a chemical bond. In this aspect, metals
may act as general acids to react with anionic and neutral ligands
(2). Metal's larger size relative to protons is compensated for by
their ability to react with more than one ligand. Metals typically
react with two, four, or six ligands. A ligand is whatever molecule
the metal interacts with. If a metal is bound with two ligands it
will form a linear complex. If the metal reacts with four ligands
the metal will be set in the center of a square that is planer or
it will form a tetrahedral structure, and when six ligands react,
the metal sits in the center of an octahedron. By clicking the
following image one can view a planar arrangement of and iron-
porphyrin system:
Slide 51
Amino acids in their peptide linkage in proteins possess groups
with the ability to bind to the metal resulting in
coordinate-covalent bonds. The free amino and carboxyl groups in a
protein can bind to the metal and this may bind the protein to a
specific, active conformation (3). The fact that metals bind to
several ligands is important in that metals play a role in bringing
remote parts of the amino acid sequence together and help establish
an active conformation of the enzyme. Zinc is the metal
incorporated in carboxypeptidase A. The zinc atom serves as a metal
ion catalyst and promotes hydrolysis. The substrate fits into the
hydrophobic pocket in carboxypeptidase A and zinc binds to the
carboxyl group of the substrate to help stabilize the
enzyme-substrate complex. In this example the zinc ion acts a
generalized acid and stabilizes the developing O- as water attacks
the carbonyl. Zinc can also perform a different role in enzymes
like the role it performs in carbonic anhydrase. Here the metal
binds H2O and makes it acidic enough to lose a proton and form a
Zn-OH group. The zinc metal serves as a nucleophile to the
substrate. Since zinc has the ability to act as an electrophile or
as the source of a nucleophilic group it is incorporated and used
by many enzymes (10).
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GENERAL FUNCTIONS OF METALLOENZYMES Hemoglobins A four-subunit
molecule, containing a iron atom in each subunit, in which each
subunit binds a single molecule of oxygen. Hemoglobin transports
oxygen from the lungs to the capillaries of the tissue. Cytochromes
Cytochromes are integral membrane proteins. Cytochromes contain
iron which serves to carry electrons between two segments of the
electron-transport chain. The iron is reversibly oxidizable and
serves as the actual electron acceptor for the cytochrome.
Phosphotransferase The Mg2+ atom serves again in electron transfer.
Alcohol Dehydrogenase A zinc metalloenzyme with broad specificity.
They oxidize a range of aliphatic and aromatic alcohols to their
corresponding aldehydes and ketones using NAD+ as a coenzyme.
Arginase The metal atom of Mn2+ is used in electron transfer.
Ferredoxin An electron transferring proteins involved in
one-electron transfer processes. Cytochrome Oxidase The copper ions
easily accommodate electron removed from a substrate and can just
as easily transfer them to a molecule of oxygen (10).
Slide 53
Regulation and Control Metalloenzyme Inhibition Approximately
one-third of the known enzymes have metals as part of their
structure, require that metals be added for activity, or are
further activated by metals. In enzymes where a metal has been
built into the structure of the enzyme molecule, the metal cannot
be removed without destroying that structure. Such enzymes include
the metalloflavoproteins, the cytochromes, and the ferredoxins. In
enzymes where metals are required to be added for activity the
metals react reversibly with proteins to form metal-protein
complexes that constitute the active catalyst. In many instances,
the complex represents a specific, catalytically active
conformation of the protein; the role of the metal appears to be
one of stabilizing that conformation (2). Because the grouping,
metalloenzymes, is so large and broad it would be almost impossible
to explain how all of them can be controlled and regulated. In
light of this, it is important to instead mention how the important
functions of metals in enzymes can be disrupted and thus inhibited.
Metals resemble protons (H+) in that they are electrophiles that
are capable of accepting an electron pair to form a chemical bond.
In doing so, metals may act as general acids to react with anionic
and neutral ligands. This characteristic of metals is helpful in
enzymatic structure and function but makes the enzyme it is part of
pH dependent. Changes in pH can disrupt this electron flow that the
metal would normally help facilitate and thus inhibit the overall
effectiveness of the metalloenzyme. Also, because of the
variability inherent to the metal's ability to react with more than
one ligand, you see metals as part of the active site in many
metalloenzymes. Competitive inhibitors in the form of
transition-state analogs are compounds believed to look like the
substrate in its transition state. To be effective the
transition-state analog must not be susceptible to reaction by the
enzyme. Competitive inhibition, via transition-state analog, has
been exhibited in the reaction of Carboxypeptidase A by a
phosphorus molecule constructed by Paul Bartlett, which can be seen
in Figure 3 (10). It functions will in inhibition of CPA because
the phosphorus atom, with its attached oxygens and nitrogen,
resembles the tetrahedral carbon atoms in the two intermediates of
Figure 2, and the transition states of all three steps (10).
Slide 54
Regulation of Metalloenzymes Through Diet As aforementioned,
metals play a large role in the activity of a multitude of
biological molecules. The source of zinc in humans is almost
entirely through diet and without the intake of metals such as zinc
in the diet one almost certainly inhibits the production and/or
activity of many vital enzymes. Among enzymes that would not be
produced by the body were it not for the presence of zinc in the
body are carbonic anhydrase, the carboxypeptidases, alkaline
phosphatase, lactic acid, and alcohol dehydrogenases. The
recommended daily dietary allowance for zinc is 15 mg., with 20 and
25 mg. During pregnancy and lactation. The average adult human
being ingests 12 to 20 mg. Of zinc per day. Deficiencies in dietary
zinc intake can result in stunted growth, enlarged liver and
spleen, and underdevelopment of genitals and secondary sex
characteristics. Outside of dietary intake deficiencies in zinc,
and thusly in enzymes that contain zinc, can be caused by the
excretion of zinc in perspiration, or by blood loss if there is
parasite infection. There is also increasing evidence that zinc
plays an important role in protein biosynthesis and utilization.
The addition of small amounts of zinc to a diet containing
suboptimal amounts of a vegetable protein, as indicated by the
growth of young rats, causes a pronounced increase in protein
utilization and growth. This defect may result from a failure in
adequate RNA synthesis. Zinc apparently inhibits the enzyme
ribonuclease. Thus, in zinc deficiency, excessive destruction of
RNA could occur. This demonstrates that the dietary intake of metal
is not only important for the production of key enzymes but also
for the inhibition of many others.