Bio Inorganic Paper III

8/3/2019 Bio Inorganic Paper III

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Paper III: Bio-Inorganic Chemistry

Some biologically important transition metals

Iron Zinc Copper Cobalt Nickel Molybdenum Human body contains 4 g

Storage & transport proteins

Ferritin, TransferrinBiological functions

Oxygen carriers Haemoglobin, Myoglobin & Hemerythrin

Enzymes Catalases, Peroxidases, Ctytochrome c oxidase

Electron transfer proteins Cytochromes, Ferredoxins, Cytochrome P450

Deficiency Symptoms Anaemia, Fatigue, Enlarged spleen

Toxicity Symptoms

Liver cirrhosis, Liver dysfunction, Siderosis, Hemochromatism, Congestive heart failure

Iron Stores Major tissue sites are liver & bone marrow/spleen Storage form: ferritin Iron storage protein 24 subunits, each 20KDa

4,500 Fe atoms are stored

Oxygen carriers

Myoglobin/Hemoglobin

Oxygen carriers Hemoglobin transport O2 from lungs to tissues

Myoglobin O2 storage protein Contain heme group


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Mb and Hb subunits structurally similar

Mb monomeric protein Hb heterotetramer (a2b2)

Myoglobin Hemoglobin

Hemoglobin

Hemoglobin (Heme + globin) Mol. Wt. : 64.5 KDa Tetramer Binds 4 O2 molecules Present in blood Transports oxygen from lungs to tissues There it transfers oxygen to myoglobin


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Myoglobin (Mb)

Monomer Present in tissues Acts as a storage reservoir for oxygen Facilitates oxygen flow within the cells Has no cooperativity effect

Myoglobin (Mb)

Mb is a compact globular protein Heme group located in crevice surrounded by non-polar residues, except for 2 histidines Non-polar residues protect Fe2+ from oxidation to Fe3+ (Hematin) which will not bind O2


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From: Stryer, LS (1988) Biochemistry (3rd Ed). New York: WH Freeman & Co.

Heme group

Heme = Fe++ bound to tertapyrrole ring (protoporphyrin IX complex) Heme non-covalently bound to globin proteins through His residue O2 binds non-covalently to heme Fe++, stabilized through H-bonding with another His residue Heme group in hydrophobic crevice of globin protein

Iron + porphyrin complex

Heme group

Corrin & porphyrin rings

In deoxy form Fe(II) is five coordinate, His as axial ligands


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Binding of oxygen in hemoglobin

Fe(II) is high spin, with ionic radius too large to fit in the cavity of porphyrin On oxygenation, low spin Fe(II) moves into the plane of the ring

Cooperativity

The oxygen binding to hemoglobin (Hb) is not independent. The presence of bound oxygen molecules favours addition ofmore oxygen molecules.


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Conversely if only one oxygen molecule is bound, it dissociates more readily than from a more highly oxygenated species. Net result is at low oxygen concentration, Hb is less oxygenated.

O2 Binding to Hb shows positive cooperativity

Hb binds four O2 molecules O2 affinity increases as each O2 molecule binds Increased affinity due to conformation change Deoxygenated form = T (tense) form = low affinity Oxygenated form = R (relaxed) form = high affinity

O2 Binding to Hb shows positive cooperativity

Oxygen Binding Curves

Mb has hyperbolic O2 binding curve Mb binds O2 tightly. Releases at very low pO2 (tissues) Hb has sigmoidal O2 binding curve Hb has high affinity for O2 at high pO2 (lungs) Hb low affinity for O2 at low pO2 (tissues)


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Oxygen Binding Curve

Oxygen Binding Curve

Bohr Effect pH sensitivity of hemoglobin is called Bohr effect Carbon dioxide released in muscle lowers the pH Increased CO2 leads to decreased pH

CO2 + H2OHCO3- + H+

At decreased pH several key amino acids are protonated. HCO3- combines with N-terminal alpha-amino group to form carbamate group.

--N3H+ + HCO3

- --NHCOO-

Carbamation stabilizes T-conformation & causes Hb to take on T-conformation (low affinity) In R-form amino acids are deprotonated, form charge-charge interactions with positive groups, stabilize R-conformation

(High affinity) The affinity of Hb to O2 decreases with lowering pH. Binding of O2 to Hb is minimum between pH 6.0-6.5 Hence, in tissues O2 is transferred from Hb to Mb


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Comparison of Hb & Mb

Hemoglobin Myoglobin

Tetramer

Carries O2, CO2 and H+

Binding of O2 is cooperative

Affinity for O2 is dependent on pH & CO2

Monomer

Carries O2

Binding of O2 is non-cooperative

Affinity for O2 is independent of pH & CO2

Oxygen carriers

Heme-containing ferrous Hemoglobin Myoglobin

Non-heme ferrous Hemerythrin

Non-heme non-ferrous Hemocyanin


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Hemerythrin

Present in lower organisms like

Peanut Worms (Phylum Sipuncula)

Hermit Sipunculan Lives in the shells of gastropods

Rock-Boring Sipunculan Associated with calcareous surfaces (coral and limestone)

Burrowing Sipunculan Burrows in fine and coarse sands

Hemerythrin

Non-heme protein with eight subunits Each subunit has two Fe atoms Coordination structure about each Fe is distorted octahedron One trigonal face is shared by the two Fe coordinated polyhedra

Both the Fe are high spin Fe(II) in deoxy form (Colorless) & low spin Fe(III) in oxy form (violet-pink)


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FeIII

OFeIII

OO

Glu

O

O

Asp

N(His)

O N(His)(His)N

(His)N

(His)N

FeII

HO

FeII

OO

Glu

O

O

Asp

N(His)

N(His)(His)N

(His)N

(His)N

OH

O2

DeoxyHr

Diferrous

OxyHr

Diferric

HydrophobicResidues

Chemistry at the Active Site of Hemerythrin (Hr)

Oxygen carrier inmolluscs,arthropodsand crustaesia, scorpions, lobster, crab Called blue blooded animals

Classes of Mollusca

Class Bivalvia (Clams, oysters) Class Gastropoda (snails, slugs) Class Cephalopoda (Squid, cuttle fish, octopus)

Haemocyanin

Non-heme, non-ferrous protein 12 sub units, each having a pair of Cu atoms Copperatoms are bound asprosthetic groupscoordinated byhistidineresidues Each pair of Cu atoms carry one O2 molecule De-oxy form has Cu(I) ions - colourless Oxyform is Cu(II)- O22- -Cu(II) - blue
http://en.wikipedia.org/wiki/Molluschttp://en.wikipedia.org/wiki/Molluschttp://en.wikipedia.org/wiki/Molluschttp://en.wikipedia.org/wiki/Arthropodhttp://en.wikipedia.org/wiki/Arthropodhttp://en.wikipedia.org/wiki/Arthropodhttp://en.wikipedia.org/wiki/Copperhttp://en.wikipedia.org/wiki/Copperhttp://en.wikipedia.org/wiki/Prosthetic_grouphttp://en.wikipedia.org/wiki/Prosthetic_grouphttp://en.wikipedia.org/wiki/Prosthetic_grouphttp://en.wikipedia.org/wiki/Histidinehttp://en.wikipedia.org/wiki/Histidinehttp://en.wikipedia.org/wiki/Histidinehttp://en.wikipedia.org/wiki/Histidinehttp://en.wikipedia.org/wiki/Prosthetic_grouphttp://en.wikipedia.org/wiki/Copperhttp://en.wikipedia.org/wiki/Arthropodhttp://en.wikipedia.org/wiki/Mollusc


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Structure of Deoxyhemocyanin

Hemoglobin vs Hemocyanin

Hemoglobin Hemocyanin

4 sub units oxygen-binding ion is iron One Fe at each active site Bluish when deoxygenated & red when

oxygenated

may be extracellular or intracellular

12 sub units oxygen-binding ion is copper Two Cu s at each active site Colorless when deoxygenated & blue

when oxygenated

always extracellularModel compounds to oxygen carriers

(Synthetic oxygen carriers)

Vaskas complex Tetraphenylporphyrin derivatives Picket fence porphyrins Capped porphyrins


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Vaska's complex

trans-chlorocarbonylbis(triphenylphosphine)iridium(I)

IrCl(CO)[P(C6H5)3]2

It has ability to bind toO2reversibly

Tetraphenylporphyrin

H2TPP is a synthetic heterocyclic compound that resembles naturally occurring porphyrins.
http://en.wikipedia.org/wiki/Vaska's_complexhttp://en.wikipedia.org/wiki/Vaska's_complexhttp://en.wikipedia.org/wiki/Oxygenhttp://en.wikipedia.org/wiki/Oxygenhttp://en.wikipedia.org/wiki/Oxygenhttp://en.wikipedia.org/wiki/Oxygenhttp://en.wikipedia.org/wiki/Oxygenhttp://en.wikipedia.org/wiki/Oxygenhttp://en.wikipedia.org/wiki/Oxygenhttp://en.wikipedia.org/wiki/Vaska's_complexhttp://en.wikipedia.org/wiki/Vaska's_complex


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Picket fence porphyrin

A picket-fence Fe(II)porphyrin complex with bound O2-

Metals, along with proteins, can harness the reactivity of oxygen by activating it and shielding it

Iron containing enzymes

Catalases Peroxidases Cytochrome c oxidase Cytochrome P-450


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Catalases

Molecular weight : 250 kDa Catalyse decomposition of H2O2 and some peroxides

H2O2 + H2O2 2H2O +O2

Peroxidases

Catalyse the oxidation of substrates by peroxides or H2O2H2O2 + SH2 2H2O+ S

Cytochrome c oxidase

The final step of the respiratory chain carries electrons from cytochrome cto molecular oxygen, reducing it to H2O.4H+ + O2 2H2O

Cytochrome c oxidase Catalyses this reaction

The three proteins critical to electron flow are subunits I, II and III. Complex IV in Fig. Contains Fe-Cu binuclear cluster

Active site of Ctytochrome c oxidase


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Electron Flow

Electron transfer through Complex IV begins when two molecules of reduced cytochrome c(top) each donate an electron to the

binuclear center CuA. From here electrons pass through heme ato the Fe-Cu center (cytochrome a3 and CuB).

Oxygen now binds to heme a3 and is reduced to its peroxy derivative (O22-

) by two electrons from the Fe-Cu center. Delivery of two more electrons from cytochrome cconverts the O22- to two molecules of water, with consumption of four

substrate protons from the matrix.

At the same time, four more protons are pumped from the matrix.Dioxygen reduction at Fe-Cu center of Ctytochrome c oxidase

Cytochrome P-450

Heme protein Present in enzymes of plants & bacteria CatalyzesR-H+ O2 R-O-H

Action of Cytochrome P-450

(a) Fe present as Fe(III) in resting state

(b) Hydrocarbon binds

(c) One electron transfer to heme

(e) Binds O2


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(f) Reduction by second electron and uptake of 2H+ ions forms Fe(IV)-oxo complex.

a) Loss of ROH and uptake of H2O

The Cytochrome P-450 Reaction CycleWhen an axial site is available on the iron porphyrin, dioxygen can bind and/or be activated there. With proton-mediated reductive

activation of the O2 molecule, a peroxo intermediate forms that converts to an FeIV=O species, the ferryl ion.

The ferryl can oxidize hydrocarbons to alcohols, epoxidize olefins, oxidize amines to amine oxides and do related chemistry.

P-450s are liver enzymes necessary for metabolism and used to convert pro-drugs and pro-carcinogens to their active forms.


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Electron transfer proteins

Cytochromes a, b, c

Heme proteinFerredoxins

Cytochrome c Structure


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Ferredoxins

Ferredoxins are small electron-transfer proteins, containing one or more Fe-S clusters Are ubiquitous in nature Participate in one-electron transfers in which one Fe atom of Fe-S cluster is oxidized or reduced Have key role in Photosynthesis and Nitrogen Fixation

Ferredoxins

Fe-S proteins present in plants & bacteria

Iron-sulfur proteins/centers

Iron is in association with inorganic sulfur atoms or with sulfur atoms of Cys residues in the protein, or both. Fe-S centers range from simple structures with a single Fe atom coordinated to four Cys -SH groups to more complex Fe-S

centers with two to four Fe atoms; (a) single Fe, (b) 2Fe-2S, or (c) 4Fe-4S centers.

Plant Ferredoxin Contains [2Fe-2S] active redox centers

Bacterial Ferredoxin Contains [3Fe-4S] and [4Fe-4S] clusters in active site


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Fe-S clusters also occur in a variety of enzymes like oxygenases, hydrogenases, nitrogenase, fumarate reductases, Sulfite reductase Succinate dehydrogenase, Xanthine oxidase In non-redox enzymes such as Aconitase

Photosynthesis

Introduction Evolution of photosynthesis Photosynthetic bodies Light-harvesting pigments Stages of Photosynthesis

Light reaction Z Scheme

Dark reaction/ Calvin cyclePhotosynthesis

Synthesis of carbohydrates from CO2 & H2O in the presence of sun light & chlorophyll 6 H2O + 6 CO2 C6H12O6 + 6 O2 Photosynthesis is a chemical process that energy from light is harvested to provide carbohydrates.

It is the major path through which carbon reenters the biosphere (from CO2).

Photosynthesis is also the major source of oxygen in the earth's atmosphere.

Evolution of photosynthesis

About 2.7 billion years ago cyanobacteria like-things evolved.


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Genetic exchange between interdependent green & purple bacteria created an organism which could live freely on the planet

wherever H2O, CO2 & light available. Tremendous increase in the biosphere

Impact on biodiversity

Photosynthesis made the atmosphere O2 rich. O2 levels increased 2.2 billion years ago due to complex eukaryotes. Cyanobacteria evolved about 2.0 billion years ago. First invertebrates about 0.7 billion years ago. First plants on land about 0.5 billion years ago. First reptiles 0.4 billion years ago.

Photosynthetic bodies

Leaves

Photosynthesis in plants occurs in leaves.- Water & carbon-dioxide enter the cells of the leaf.

- Sugar & oxygen leave the cells of the leaf.

Water transported to the leaf through the xylem. Stomata (plural for stoma) provide a pathway for carbon dioxide to be taken up & oxygen to be released. Mesophyll cells fill the region between the epidermis layers & contain the chloroplasts.

Chloroplasts & Thylakoids

Chloroplasts are organelles specific to plants.- Approximately 4-10 mm diameter.

Contain:- The stroma (the matrix within the inner membrane).

- Flattened vesicles called Thylakoids.


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Thylakoid membrane

-Thylakoid membrane has two distinct regions.

- Stacked regions called grana which contain photosystem II.

- Non-stacked regions, called stroma lamellae, which contain photosystem I & ATPsynthase.

Light-harvesting pigments

The major light absorbing pigment on thylakoid membrane is chlorophylls Chlorophylls (aand b) resemble the heme group of hemoglobin, except that the central Fe2+ is replaced by a Mg 2+


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Structure of Chlorophyll

Chemistry of Photosynthesis

In photosynthesis Carbon dioxide is reduced to glucose. The electrons needed for this reduction come from water. The energy needed for this reduction comes from light (ATP, NADPH).

Stages of Photosynthesis

Two stages of photosynthesis Light reaction - photolysis of water Dark reactioncarbondioxide fixation Both the reactions take place in chloroplast

The Two Reactions The light reactionsrequire light, which is converted to chemical energy & conserved as


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high energy compound ATP reducing power of NADPH

The light-independent dark reactionsoccur either in the light or in the dark.NADPH and ATP produced by the light reactions are used in the reductive synthesis of carbohydrate from CO 2 and water

The light reactions of photosynthesis stop when the sun goes down. However, CO2 fixation can continue as long as ATP and

NADPH are available.

The Dark Reactions

6CO2 + 12H2O + 18ATP + 12NADPH 6C(H2O) + 6O2 + 18 ADP + 18Pi + 12 NADP + 6H2O

The Light Reactions

Classes of reaction centres

Photosynthetic bacteria, algea & plants fix CO2.- Produce 10 billion tons of carbohydrate annually.

- Eight times human energy consumption.

Two types of reaction centre:- Type-I (green sulphur bacteria) use iron-sulphur centres as terminal electron acceptor.

- Type-II use (purple photosynthetic bacteria) use quinones as terminal electron acceptor. In cyanobacteria, algea & plants a more complex system.

Tools of Photosynthesis

Antenna Complexes

PS I

PS II

Cytochrome B6/F Complex

Oxygen Evolving Complex

ATPase

Antenna Complexes -The two antenna complexes (one for each Photosystem) contain Chlorophyll, accessory pigments, and

proteins. They collect radiant Energy to excite rxn center chlorophylls.PS I - PS I a complex of molecules, with an Antenna complex, Proteins, Ions, a molecule called phylloquinone, a reaction center

chlorophyll (called P700), and Ferredoxin. Ferredoxin is an iron-containing molecule that passes an excited electron to NADP+.

PS II - PS II is a lot like PSI. It contains proteins, pigments, metal and other ions, Plastoquinones, Pheophytin, and a special reaction

center chlorophyll molecule, called P680.

Cytochrome B6/F Complex- The cyt b6-f complex contains proteins, metal ions and a special iron-sulfur protein. It also

translocates protons across the Thylakoid membrane, much like the etc.

Oxygen Evolving Complex - The OEC is part of PS II. It contains several Mn and Fe containing proteins which oxidize water (a


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The roles of PSI and PSII

PS I produces reducing powered NADPHPS II uses light energy to drive two chemical reactions - the split of water producing O2 and releasing electrons into an electron

transport chain (Photosynthetic Electron Transport).

Photosystem I

- Accepts electrons from plastocyanin.- Reduces ferredoxin, an Fe/S protein.- NADP+ is reduced to NADPH from ferredoxin by ferredoxin-NADP+ oxidoreductase.

Photosystem II

- Requires l < 680 nm.- Abstracts electrons from water & raises them to sufficiently negative potential so as to reduce plastoquinone (PQ).

Structure of PSII (from a cyanobacterium)

The primary electron donor is P680 Formed by two chlorophylls 10 apart. Contains two further chlorophylls, pheophytin & bound plastoquinone sites. Contains a 4Mn complex which abstracts electrons from water.

Active site of PSII

Five metal ions in the active site.- 4 manganese ions &- 1 calcium ion.


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- 3 Mn & 1 Ca at four corners of a distorted cube.- Oxygen atoms at the other corners.

- The fourth Mn ion is liganded by one oxygen of the cube.

Water splitting reaction

The photo-excited P680+ is reduced by a tyrosine residue, Tyrz.

Tyrz+ in turn abstracts an electron from the Mn cluster. Four photon absorption steps lead to 4Mn being oxidised to 4Mn+.

- Highly electropositive.- Spontaneously accept 4 electrons from H2O (Em,7 of the O2/2H2O couple is 810 mV).- Most electropositive reaction in nature.

Electron flow in PSII

Electron from photo-excited P680 flows to QA (& then to QB) via a chlorophyll and a pheophytin. Second electron transfer releases a quinol from QB. After each charge separation step P680+ abstracts one electron from a nearby manganese cluster via a tyrosine residue (Tyrz). Four positive charges accumulate on the Mn cluster which oxidise two water molecules & release O2 & 4H+.

Water splitting reaction

The enzyme accumulates four positive charge-equivalents.

Deprotonation occurs to compensate the charge accumulation on some steps, before oxidizing 2H2O and releasing O2.

The valence of the Mn ions increases on the S0 to S1 to S2 steps; Less certain for the S3 & S4 steps.


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Cytochrome B6/F Complex- contains proteins, metal ions and a special iron-sulfur protein.

It translocates protons across the Thylakoid membrane.

It accepts electrons from PQ & passes them on toplastocyanin(like cytochrome c of the mitochondria).

The Z scheme of photosynthetic electron transport

Light driven electron flow from H2O through PS II Electron transfer within the cytochrome b6/f complex Electron transfer from the cyt b6/f complex to PSI

e- acceptor

lightNADPH

NADP+

electrontransportsystem

ATP

H2O 2e- + 2H+ + O

e- acceptor

P680 antennacomplex

P700 antenna

complex


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Light driven ATP synthesisphotophosphorylation

Photophosphorylation is represented by the Z scheme, where electrons activated by photons at PSII and PSI flow from H2Oto NADP+ and a H+ gradient is established by cytb6/f complex to drive ATP synthesis.

ATP and NADPH produced during light reaction, are consumed by the carbon (dark) reactions, which reduce CO 2 to carbohydrate

([CH2O]n).


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Carbon (Dark) Reactions

The Calvin Cycle

Also known as photosynthetic carbon reduction cycle, or reductive pentose phosphate (RPP) cycle.


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The Calvin Cycle

Ribulose Bisphosphate Carboxylase/Oxygenase (RuBisCO)

The only enzyme capable of fixing CO2.

- Attaches CO2 to ribulose bisphosphate.- Clips the lengthened chain into two identical phosphoglycerate pieces.

Active site of Rubisco

Arranged around a magnesium ion (green). The magnesium ion is fixed by three amino acids, including a modified lysine (an extra

CO2 is attached).

The enzyme which possesses both oxygenase and carboxylase activity, represents ~40% of the total soluble protein of mostleaves.


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Balancing the photosynthesis equation

Light driven reactions:

12 NADP+ + 18 ADP + 18 P + 6 H+ + 48 hn

6O2 + 12 NADPH + 18 ATP + 6H2O

Dark reactions:6CO2 + 18 ATP + 12 NADPH +12 H2O

C6H12O6 +18 ADP + 18Pi + 12 NADP+ 6 H+

Sums to give overall: 6 H2O + 6 CO2 + 48 hn C6H12 O6 + 6 O2


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Biological Nitrogen Fixation

Definition Significance Types of nitrogen fixation

atmospheric fixation industrial fixation biological fixation

Nitrogen cycle Mechanism of N-fixation

Nitrogen fixation

Conversion of atmospheric nitrogen into soluble ammonium or nitrate ions is called Nitrogen fixation.

Significance of Nitrogen

Often a limiting nutrient for algal growth Occasionally Toxic

Types of Nitrogen

N2 gas Very Abundant, mostly unavailable DIN, Dissolved inorganic nitrogen

NH3 / NH4

+

Ammonia / Ammonium (nutrient, toxic at high levels) NO3- / NO2- Nitrate / Nitrite (nutrient, toxic at high levels) Organic nitrogen

PON Particulate organic nitrogen (e.g. algae, bacteria, detritus) DON Dissolved organic nitrogen (proteins, tannins, etc. etc.)

Nitrates are essential for plant growth

Nitrogen is a critical part of amino acids, nucleotides and other biomolecules

Nitrogen Fixation

Three processes are responsible for most of the nitrogen fixation in the biosphere:

Atmospheric fixation Industrial fixation Biological fixation


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Nitrogen from the atmosphere

Atmospheric nitrogen fixationThe enormous energy of lightning breaks nitrogen molecules and enables their atoms to combine with oxygen in the air forming

oxides of nitrogen NOx. These dissolve in rain, forming nitrates, which are carried to the earth.

Atmospheric nitrogen fixation contributes some 5-8% of the total nitrogen fixed.

Industrial N-Fixation

The Haber-Bosch ProcessN2 + 3H2 2NH3 - 92kJ

The Haber process uses an iron catalyst High temperatures (500C) High pressures (250 atmospheres)

The energy require comes from burning fossil fuels (coal, gas or oil) Hydrogen is produced from natural gas (methane) or other hydrocarbon Ammonia can be used directly as fertilizer, but most of it is further processed to urea and ammonium nitrate (NH4NO3).


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Ammonification

Nitrogen enters the soil through decomposition of protein in dead organic matterAmino acids + 11/2O2 CO2 + H2O + NH3 + 736kJ

This process liberates a lot of energy which can be used by the saprotrophic microbesNitrification

This involves two oxidation processes The ammonia produced by ammonification is an energy rich substrate forNitrosomasbacteria. They oxidise it to nitrite:

NH3 + 11/2O2NO2

- + H2O + 276kJ

This in turn provides a substrate forNitrobacterbacteria to oxidise the nitrite to nitrate:NO3

- + 1/2O2NO3- + 73 kJ

This energy is the only source of energy for these prokaryotes. Thus, they are chemoautotrophs.Biological nitrogen Fixation

This accounts for most of the fixation of atmospheric N2 into ammonium. Scientist estimate that biological fixation globallyadds approximately 140 million metric tons of nitrogen to ecosystems every year.

This is performed exclusively by prokaryotes using the enzyme Nitrogenase. Most of these bacteria are free living in the soil, a few form symbiotic associations with higher plants.

The prokaryote directly provides the host plant with nitrogen in exchange for other nutrients and carbohydratesN- Fixation Requires Anaerobic Conditions

As oxygen irreversibly inactivates the nitrogenase enzymes involved in nitrogen fixation, nitrogen must be fixed underanaerobic conditions.

Hence, each of the N-fixing organisms either functions under natural anaerobic conditions or can create an internalanaerobic environment in the presence of oxygen.


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Types of Biological Nitrogen Fixation

Free-living (asymbiotic)

Cyanobacteria Azotobacter

Associative

RhizosphereAzospirillum Lichenscyanobacteria Leaf nodules

Symbiotic

Legume-rhizobia Actinorhizal-Frankia

Cyanobacteria

only photosynthetic prokaryotes live in water environments colonial and solitary some perform nitrogen fixation free-living photo-autotrophs

N- Fixation by cyanobacteria

Cyanobacteria can fix nitrogen under anaerobic conditions such as those that occur in flooded fields In Asian countries, nitrogen fixing cyanobacteria are the major means of maintaining an adequate nitrogen supply

in rice fields They fix nitrogen when the fields are flooded, and die as the fields dry, releasing the fixed nitrogen into

the soil

Thus cyanobacteria are essential to maintain the fertility of semi-aquatic environments like rice paddies.Types of N-Fixing bacteria

Some nitrogen-fixing bacteria (Rhizobia) live in symbiotic relationship with plants of legume family (e.g., soybeans, alfalfa). Some establish symbiotic relationship with plants other than legumes (e.g., alders). Some live free in the soil (Non-smbiotic)


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Azotobacteraerobic Clostridium - anaerobic

The nitrogen fixers

The most common association is between members of the plant family leguminosaeand bacteria of the generaAzorhizobium. Rhizobiumbacteria grow in root nodules.

Azotobacterare bacteria associated with the rooting zone (the rhizosphere) of plants in grasslands. The most familiar examples of nitrogen-fixing symbioses are the root nodules of legumes (peas, beans, clover, etc.) Members of the bean family (legumes) and some other kinds of plants form mutualistic symbiotic relationships with nitrogen

fixing bacterial.

In exchange for some nitrogen, the bacteria receive from the plants carbohydrates and special structures (nodules) in rootswhere they can exist in a moist environment.

Legume + RhizobiumTeam

Legume PlantRhizobiumbacteria

- forms a nodule in response to Rhizobium- provides energy and protection for the bacteria

in the nodule

- converts fixed N to organic N and produceshigh protein forage

- infects plant- provides genetic information that allows N

fixation

- uses the plant energy and nodule environment toaccomplish N fixation

Legume plants and Rhizobiumbacteria team up to remove N from the air Nodules form on legume roots when this system is working

Mechanism of N-Fixation

Nitrogen molecule (N2) is quite inert. To break it apart so that its atoms can combine with other atoms requires the input of substantial amounts of energy. Hence, biological nitrogen fixation requires a complex set of enzymes and a huge expenditure of ATP.

Why does nitrogenase need ATP?

N2 reduction to ammonia is thermodynamically favorable However, the activation barrier for breaking the N-N triple bond is enormous 16 ATPs provide the needed activation energy


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N N stable triple bond

Its activation is a very energy-demanding process

Six electrons must be transferred and the process occurs in several steps.N2 +16 ATP + 8H

+ +8e-2NH3+16 ADP+16Pi + H21 e/2ATP per cycle

2ATP binding shifts reduction potential

Fe-S clusters

Iron

Molybdenum

8 electrons (6 for N2, 2 for H2)

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Bio Inorganic Paper III