BIO-INORGANIC CHEMISTRY

BIO-INORGANIC CHEMISTRYSYLLABUS:Elements of life: Essential major, trace and ultratrace elements.Basic chemical reactions in the biological systems and the role of metal ions (specially Na+, K+, Mg2+, Ca2+, Fe3+/2+, Cu2+/+, and Zn2+).Metal ion transport across biological membrane Na+-ion pump, ionophores. OXYGEN CARRING PROTEINS1. Hemoglobin(Hb)2. Myoglobin(Mb)3. Hemerythrin4. HemocyaninELECTRON TRASPORT PROTEINS1. The iron-sulpher proteins: Ferredoxins.2. A group of heme proteins: Cytochromescarbonate bicarbonate buffering system and carbonic anhydrase.Biological nitrogen fixation: Nitrogenase enzymePhotosynthesis: Photosystem-I & Photosystem-II.

Toxic metal ions and their effects, Chelation therapy: Pt and Au complexes as drugs. Metal dependent diseases.

Elements of lifeThe evolutionary processes have selected about 30 element as essential elements, out of 92 naturally occurring ones, to perform one or more of the physiological functions and have rejected the others.What is essential elements?Essential elements are those in absence of which growth is inhibited.Nature always selects elements of small atomic numbers(why?)

Almost 99% of the mass of the human body is made up of six elements:what are these elements?1.Oxygen2.Carbon3. Hydrogen4.Nitrogen5.Calcium6. phosphorus.

Only about 0.85% is composed of another five elements:potassium,sulfur sodiumchlorine andmagnesium. All are necessary to life.

Heavy elements are usually toxic.In terms of weight percent 11 of these 30 essential elements are considered as major elements and the rest of as trace elements.

MAJOR ELEMENTS

The elements which are present in bulk are called major elements, as they are required in the living body in relatively large amounts. Six elements of organic matter.And Na, K, Mg, Ca, Cl, five monoatomic ions ,I,e; total 11 elements are collective known as major elements.Trace ElementsTrace elements are present at low levels in organisms and make up just 0.5% of living cells. However, living things would not be able to survive without trace elements. Trace elements include: Iron(Fe) Iodine(I) Manganese(Mn) Molybdenum(Mo) Selenium(Se) Silicon(Si)Tin(Sn)Vanadium(V) Boron(B) Chromium(Cr)Cobalt(Co) Copper(Cu) andFluorine(F).

Iron is found in red blood cells and helps to carry oxygen in the blood stream. Iodine is important for making different forms of thyroid hormone, which regulates growth and energy levels in humans. Many of the trace elements are required by enzymes in order to make chemical reactions happen.

Ultra-trace elementsThe elements which presents in exceedingly small amounts are called Ultra-trace elements.For example: Cd, As, Pb, Ge, Li, Rb, Ba, Br.,etc.STORAGE AND TRANSPORT OF METABOLIC ENERGYThe living cell requires large amount of fuel to sustain its activity. Majority of the reactions in the cells are endergonic and non-spontaneous.Spontaneous cell decomposition takes place upon death.

Adenosine triphosphate (ATP) is theenergycurrency for cellular processes. ATP provides the energy for both energy-consumingendergonicreactionsand energy-releasingexergonicreactions, which require a small input ofactivation energy. Metabolic energy is stored as the P-O bond energy of nucleotides, particularly ATP.

An amount of energy equivalent to 7.3 k.cal.mole-1 is required to phosphorylate ADP with inorganic phosphate to produce ATP.

When the chemical bonds within ATP are broken, energy is released and can be harnessed for cellularwork. The more bonds in amolecule, the morepotential energyit contains. Because the bond in ATP is so easily broken and reformed, ATP is like a rechargeable battery that powers cellular process ranging fromDNAreplication toproteinsynthesis.

Mg-ATP and Mg-ADPIn the biological Ph(7-7.5) ATP and ADP exist as highly charged anionic species, viz. ATP4- and ADP3-.Due to high affinity of triphosphate and pyrophosphate groups for bivalent metal ions and relatively high concentration of Mg2+ ion in the intracellular fluid in intact living cells, ATP4- and ADP3- ions predominantly occur in the forms of their Mg2+ complex, viz. Mg-ATP2- and Mg-ADP-The affinity of ATP4- for Mg2+ ion is about 10 times stronger than that of ADP3-.

ATP is an unstable molecule which hydrolyzes to ADP and inorganic phosphate when it is in equilibrium with water. The high energy of this molecule comes from the two high-energy phosphate bonds. The bonds between phosphate molecules are called phosphoanhydride bonds. They are energy-rich and contain aGof -30.5 kJ/mol.

In the absence of metal ions, non-enzymic hydrolysis of ATP is first order in the ATP species.

Metal ions catalysed hydrolysis of ATP is first order in M2+ ions (M= Mg, Cu, Zn,Mn, Ni) and first order in the ATP species.

Reactivity's of the M2+ ions fall in the order : Mg2+ Mn2+ Zn2+ Cu2+ which is almost parallel to the stability order of M2+ -APT complexes.

Hydrolysis of ATP

Removing or adding one phosphate group interconverts ATP to ADP or ADP to AMP. Breaking one phosphoanhydride bond releases 7.3 kcal/mol of energy.

ATP+H2OADP+Pi G = -30.5 kJ/mol

ATP+H2OAMP+2Pi G = -61 kJ/mol

2ADP+H2O2AMP+2Pi G = -61 kJ/mol

At pH 7,

ATP4+H2OADP3+HPO42 + H+

Mechanism of ATP hydrolysisEntropy of activation is negative in all these cases, indicating an associative (SN2) path.The transition is a 1:1 metal:ATP complex on which a solvent H2O molecule makes a nucleophilic attack to produce the metal-ADP complex and H2PO4-

Faster rate of hydrolysis in presence of M2+ ionsRate of hydrolysis of ATP in the presence of M2+ ions is much faster than in absence of metal ions(why?)

This is because, metal ions coordination to the phosphate groups lowers the negative charge of the ATP species by two units.

This favours nucleophilic attack by H2O molecules on the terminal phosphorous atom.

Rate of hydrolysis of Mn2+ -ATP and Cu2+-ATPThough Mn2+ -ATP and Cu2+-ATP complexes have comparable stabilities, Mn2+ ions catalysed hydrolysis of ATP is much slower than the Zn2+ ions catalysed reaction.This is because, Zn2+ ion binds to and phosphate groups of ATP only, while, Mn2+ ion possibly binds to , and -phosphate groups.Thus, all the the P-O bonds are polarised in the Mn2+-ATP complex. This results in an overall lower polarisation of the individual P-O bonds, hence the slower rate of hydrolysis. Why is ATP hydrolysis an exergonic reaction?

Theentropy, which isthe level of disorder,of ADP is greater than that of ATP. Therefore, due tothermodynamics, the reaction spontaneouslyoccurs because it wants to be at a higher entropylevel. Also, theGibbs' free energyof ATP is higher than that of ADP. Naturally, molecules want to be at a lower energy state, so equilibrium is shifted towards ADP.Electrostatic repulsion:of the four negative charges on theoxygensof the ATP molecule. Naturally, like charges repel and opposite charges attract.Therefore, if there are four negative charges in close proximity to one another, they will naturally repel each other. This makes ATP a relatively unstable molecule because it will want to give away its phosphate groups, when given the chance, in order to become a more stable molecule.

Creatine-Phosphocreatine interconversion and ATP

Creatine is a biochemically important amino acid derivative.over 90% of the Creatine in the body of an adult is present in the msucles.

Its concentration in blood and body fluid is normally very low reaches high values values under renal dysfunction.

Creatine, whether synthesized naturally in the body or supplemented by dietary sources, is important for energy production in the human body. The initial energy needed for muscle contraction is provided by the molecule ATP.ATP can only supply enough energy for a few seconds of muscle movement, so in order for sustained muscle contraction to occur, ATP must be regenerated. This is where creatine comes in. In skeletal muscle, creatine exists in equilibrium with its phosphorylated form, which is called creatine phosphate or phosphocreatine.

One third of the creatine in skeletal muscle is free creatine, and the remaining two thirds is phosphorylated.

Phosphocreatine can give up its phosphate group to the ADP molecule, resulting in the regeneration of ATP.

According to Le Chteliers Principle, as the concentration of ATP is depleted in the first few seconds of intense exercise, the phosphocreatine-creatine equilibrium shifts to favor the formation of ATP. ATP can then be used again to power muscle contraction for up to 10 seconds of extremely intense activity, such as a 100-meter sprint.

FuctionsPhosphocreatine can anaerobically donate a phosphate group toADPto formATPduring the first 2 to 7 seconds following an intense muscular or neuronal effort.

Conversely, excessATPcan be used during a period of low effort to convertcreatineto phosphocreatine. The reversible phosphorylation of creatine (i.e., both the forward and backward reaction) is catalyzed by severalcreatine kinases. The presence of creatine kinase inblood plasmais indicative of tissue damage and is used in the diagnosis of myocardial infarction.

The cell's ability to generate phosphocreatine from excessATPduring rest, as well as its use of phosphocreatine for quick regeneration ofATPduring intense activity, provides a spatial and temporal buffer ofATP concentration.

In other words, phosphocreatine acts as high-energy reserve in a coupled reaction; the energy given off from donating the phosphate group is used to regenerate the other compound - in this case,ATP.

Phosphocreatine plays a particularly important role in tissues that have high, fluctuating energy demands such as muscle and brain.

29OXGEN TRANSPORT PROTEINSNature has designed four O2-carrying proteins for transport and storage of oxygen in biological systems.These are:

1. Hemoglobin(denoted as Hb)and2. Myoglobin(Mb) are dioxygen (O2) binding metalloproteins containing an iron porphyrin system, Fe(II)-heme proteins.

Hemoglobin is present in Red Blood Cells (RBC) and helps in transport of dioxygen from lungs to tissues. Whereas, myoglobin stores dioxygen and is present in muscles.

3. Hemerythrin, a non -heme Fe(II) proteinsOccurring in several marine invertebrates and lower forms of life. Its main function is O2-storage.

Active site of hemerythrin before and after oxygenation.4. HemocyaninHemocyanine are copper containing oxygen transport proteins, occuring in a number of invertibrate, viz., snail, quids, cuttle, octopus etc.

HEMOGLOBIN & MYOGLOBIN

ACTIVE SITE STRUCTURE

Mb is a monomeric protein (MW = 17,100daltons) having a single poly peptide chain that is not conductive of self association. On the other hand, Hb is a tetrameric protein (MW= 64500daltons), consisting of two and two peptide chains interlinked trough hydrogen bonded (COO-..NH3+ ) interactions.X-ray study showed that the disappearance of these salt bridge bonds oxygenation.

The active site of both Hb and Mb contains the heme group in which Fe(II) is equatorially coordinated by the four pyrrole nitrogen atom of protoporphyrin IX.The 5th position is coordinated by the imidazole nitrogen atom of histidine of the chain(i.e., the globin).The 6th position in deoxy-Hb or deoxy-Mb is vacant, but hdrophobically shielded by the protein chain.As a result, only non polar neutral molecules such as o2 CO, etc. can bind to the sixth position.In the absence of the protein (globin), the 6th position is irreversibly oxidised by oxygen of the air to Fe(III)- heme, hematin.The latter, because of its residual positive charge, is reluctant to bind uncharged ligands such as O2 , but readily binds charged liands such as CN- , S2- , OH- etc.; which inhibit the oxygenetion.

BIOLOGICAL IMPORTANCE OF Hb&MbHemoglobin(Hb) and Myglobin(Mb) are responsible for the transport and storage of oxygen in higher animals. Viz., mammals. Hb transports oxygen from its source(vis., lungs, skin and gills) to the site of its use inside the muscle cells, where oxygen is transferred to Mb for use in mictrochondrial oxidatio9i.e., respiration.Role of Distal(E7)and Proximal(F8) Histidine Residues in Hb & MbIndeoxy-hemoglobin, four of the coordinated sites of iron are occupied by nitrogens of porphyrin ring. The fifth site is occupied by Histidine residue (called proximal histidine) of globin. The sixth position is occupied by weakly bonded water molecule. Hence some authors tend to report Fe(II) ion in deoxy form as pentacoordinated. Deoxy-hemoglobin is said to be in T-state (tense).On the opposite side of the proximal histidine,there is one more histidine group (called distal histidine) placed near the iron ion. It forces the binding of dioxygen in "end on bent" confirmation.

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Function of proximal Histidine Residues(F8)The proximal histidine (F8) residue acts as a good -donor to facilitate the central metal to act as a better -donor towards the -acid ligand(e.g.O2)at the trans-position.

This largely helps O2 to acts as a better -acid ligand(i.e. -acceptance) to induce the spin at iron, i.e. O2 acts as a relatively strong field ligand. If the base(B) is itself a good -acid ligand, then formation of the O2-adduct will be disfavoured. CO is a powerful poison to Hb and Mb,as the heme group has very high affinity for the -acid ligand like CO.Function of Distal Histidine Residues(E7)Presence of distal histidine residue in the region of sixth coordination site, it does not allow CO to form the linear Fe-CO bond and CO is forced to make a bent bond.The bent conformation discourages the binding of CO to heme iron. Otherwise, CO may have even more affinity with the iron ion. It is observed that CO binds to hemoglobin 200X stronger than dioxygen but binds 20,000X stronger with unprotected heme.Thus the distal histdine (protein) weakens the interaction with CO and optmises the binding of O2 in Hb and MbAdditionally the imidazole moity of distal histidine stabilizes the oxygenated compound through H-bonding. Nature of bondingThe bonding mechanism of oxy-Bb and oxy-Mb can be explain by considering simplest possibilities:Coordination of singlet O2 to low spin Fe(II); O2 as a one electron acceptor leading to low-spin Fe(III) and O2- (Superoxide).In the de-oxy form, if square pyramidal geometry is consider the the electonic configuration of high spin Fe(II)In the oxy-condition, if FeIII O2interaction is considered then the electronic configuration of low-spin FeIII is t2g5(assuming octahedral geometry)

If FeII(L.S)-singlet O2 interaction is considered then then the electronic configuration of low spin FeII is t2g6.Oxy Hb/Mb is paramagnetic/diamagnetic?In the model system, FeIII O2- the unpaired electron(t2g5 ) of FeIII undergoes anti-ferromagnetic coupling with the unpaired electron in g* of O2-.Thus both the models explain the diamagnetic character of oxygenated heme unit.

Posing of Hb and MbDifferent -acid ligands like CO, NO,PF3 which are electrically neutral and not much bulky can compititively replace O2 from the sixth octahedral site of Hb & Mb.Consequently the O2 transport mechanism gets arrested and toxic effect arises. CN- may also bind the site but due to presence of heme pocket surrounded by hydrophobic environment does not welcome CN- easily.CO affinity of Hb and Mb is drastically diminished due to the steric hindrance by the distal histidine (E7).But in the industrial pollution and automobile exhaust produce a large amount of CO which on inhelation produces carboxyhemoglobin(HBCO).

This is why, it is now almost mandatoty to use catalytic convertersinto the exhaust system to convert CO and NO int CO2 and N2 respectively.Recent report demend that ZnO + CuO catalyst can effectively convert CO into CO2 .It is very interesting that the posoning effect of CO is not cumulative, as it can be replaced by O2 in fresh air.

ALLOWABLE CONCENTRATION OF COThe maximum allowable concentration of CO in air is 50 ppm for working 8 hours.Working of 4 hors in the atmosphere of 300ppm CO, leads to blocking of 35% of Hb. Working of 4 hors in the atmosphere of 800ppm CO, leads to blocking of 60% of Hb.During cigarette smoking, CO concentration may go upto 200-400 ppm and in a heavy smoker 5-15% of Hb remain as HbCO.

Detoxification of Hb & MbSome oxidising agent such as nitrite, chlorates, etc. can oxidise Fe(II) present in hemoglbine and myoglobin giving rise to methemoglbin (Met-Hb) i.e. ferrihemoglobin and metmyoglobin (Met-Mb) respectively which are not able to transport O2.

Why CN- is deadly toxic?CN- actually blocks the cytochrome c oxidase involved in the respiratory chain.

Treatment:To remove the bound CN- from cytochrome c oxidase, some methemoglobins (Met-Hb) are to be generated either by inhalation of amylnitrite vapour or by injection of NaNO2 solution. (Met-Hb) bearing Fe(III) can bind CN- more strongly than the cytochrome c oxidase. Consequently, CN- is removed from the respiratory chain to regenerate the electron tunneling path.Presently Co(II)-edta comlpex is also therapeutic use.

Acid-base balance and HbBoth the deoxy and oxy-forms of hemoglobin can acts as weak Bronsted acids. Hence in the presence of corresponding cojugate base, it can acts as a buffer to restore the biological Ph as the corresponding pKa values lies close to 7.0.

Besides this, hemoglobin plays an active role in transporting carbon dioxide from the tissues(i.e. sites of generation of CO2) to lungs. Electron Transport ProteinsElectron transport proteins are responsible for the transport of reducing equivalents (electrons) from a biological redox couple having a lower standard redox potential to one having a higher standard redox potential.standard redox potential of an electron transport protein should be intermediate between those of the electron acceptor and the electron donor couples.Electron transporting mettallo proteins are mainly the 1. Iron-sulpher proteins, viz.,Ferredoxins2. The Fe(III)-heme proteins, viz., cytochromes.Both this groups operate through their Fe(III)-Fe(II) couples.

Cytochromes Cytochromes are a group of Fe(III)- heme proteins that function as electron carriers in mitochondrial oxidation, photosynthesis etc.They are classified as a, b, c, etc. depending on their absorption spectra, their porphyrin substitution and in iron coordination.Fe in cytochromes is equatorially coordinated by four pyrrole nitrogen atoms of the porphyrin ring system. 5th amd 6th positions of iron are axially coordinated by different groups of the protein chain. Active site structure of cytochrome cThe active site of cytochrome c is the heme group which is covalently bonded with the polypeptide chain of 104 amino acid residues.Fe remains octahedrally coordinated in both the oxidised and reduced forms.One axial position is coordinated by the imidazole Natom of histdine-18 and the other axial position is occupied by the thioether S atom of methionine-80.Three dimentional shape of the protein chain looks roughly sperical. The heme group is surrounded by a hydrophobic polypeptide chain.

Electron transferRate of electron transfer with cytochrome c is 103 times slower than the rate of electron transferwith analogous compounds of iron.E.g., kobs (sec.-) for cytIII/ cytII is 5x104, while for FeIII(phen)3/FeII(phen)3 it is 1x107.Slow rate of electron transfer indicates that the heme c is buried in a hydrophobic protein pocket and only an edge of the prosthetic group is near to the surface.Electron transfer occurs through this exposed heme edge and involves the

Why slow rate of electron transferDue to hydrophobic wrapping of the heme group by the protein chain, the electron donating groups fails to make direct contact with the ultimate electron accepting site (i.e., FeIII in heme c), hence slow rate of electron transfer.Fe in both the oxidised and reduced forms of cytochrome c is in low spin configurations which are favourable for outer sphere electron transfer.

Significance of cyt.c There is a great significance of cyt.c in the evolution process. Cyt.c is the oldest chemical compound evolved more than 1.5 years ago and it is widely distributed in the biological world. The different cyt.c from different sources mainly differ in the amino acid sequence of the polypeptide chains.

In fact, a family tree of the evolution process from lower animal to higher animal can be constructed in terms of the amino acid sequence of the protein chain in cyt.c.

Iron-sulphur proteinsIron-sulphur proteins function as electron carriers in biological redox reactions, viz., photsynthesis, nitrogen fixation and mitochondrial respiration.These consist of non-heme iron, coordinated by cysteine sulphur(-SH) and acid labile inorganic sulphide sulphur(S2-).Iron-sulphur proteins are often abbeaviated as n-(Fe-S*) centres, where, n stand for the number of iron cations per protein and S* stands for the inorganic sulphide sulpher (S2- ) usually n in number.These labile sulphur atoms are liberated as H2S on acidificationof the proteins and are readily air oxidised to elemental sulphur.

Iron in these proteins are approximately tetrahedraly coordinated by four sulpher atoms, of which at least one is cysteine sulpher from the protein chain.

The electron transpot by ferredoxins take place via Fe(III)/Fe(II) couple but the redox potential of Fe-S proteins are close to that of the H+/H2 couple and vary widely depending upon the nature of the protein environment around iron(where as redox potential of aqueous Fe(III)/Fe(II) is 0.785v).

E.g., E0 of ferredoxine in photosynthetic bactrium, chromatium = -0.49 but the E0 of ferredoxine in beef-heart mitochondria = +0.22v.Ferredoxins,2Fe-2SOccurrence: 2Fe-2S Ferredoxins occur in the chloroplasts of many plants, in several bacteria, beef-heart mitochondria and in pig adrenal glands.Active site structure: These consist of a single poly peptide chain of 98 amino acids (MW 10,500 daltons).Its active site contains two iron centres bridged by two acid labile (S2-) sulphur and each Fe is bound to two cysteine sulphur atoms of the protein chain in such a maner that the individual (cys-S)2Fe(S2-)2 units appear tetrahedral, providing high spin configuration to iron. Mechanism of electron transferOxidised form: In the oxidised form, both the iron atom ar e in Fe(III) state with high spin( t2g4eg2 , S = 5/2) configuration.Yet the protein is diamagnetic and is e.p.r. silent due to antiferromagnetic FeIII ..FeIII coupling.The dz2 orbital of each iron makes a overlap with the orbital of S2- ions. The dxz and dyz orbitals of iron make -overlaps wiyh the vacant d orbitals of the two cysteine sulphur atoms.the dxy orbital, of iron atom remain non-bonding.The bridging sulphide (S2-) ligands enable the individual paramagnetic Fe(III) centres to pair up with each others spin throug super-exchange interaction. The reduced form of 2Fe-2SThe reduced form of the protein is paramagnetic for one unpaired electron and is e.p.r. active(gI=1.97). Which corresponds to reduction of one of the two Fe(III) centres to Fe(II).

2Fe-2S ferredoxins, therefore, function as one-electron transport proteins.

In the reduced form, a high spin FeIII (S= 5/2) and a high spin FeII (S=4/2) are anti-ferromagnetically coupled to give a net electron spin(S=1/2) in the ground state.

Electron transport occurs with very small energy transfer, as the redox potential (E0) of this protein is very low(e.g. -0.2to -0.4v).Iron centre in the reduced form are non-eqivalent, though they are equivalent in the oxidised form. Due to reduction, the ionic radius changes from 0.63A0 in high spin Fe(III) to 0.77A0 in high spin Fe(II).This distort the planarity of Fe (S2-)Fe moity and initiates non-valence(tertiary and quaternary) interactions in the proein chain.Conversely, alteration in the protein conformation may alter the redox potential at the avtive site of these proteins. 4Fe-4S, FerredoxinsThese proteins can undergo one electron redox reactions, though their biochemical functions are less well known.

These are not generally classed as ferredoxins, rather, these are often called high potential fron proteins and are abbreviated as HIPIP.

Active site of these proteins consist of four iron atoms, four acid labile sulphide sulphur(S2-) and four cysteinyl sulpher atoms arranged in a cubic structure.Mode of bonding

Mode of bonding of iron and sulphur(S2-) in 4Fe-4S proteins is the same as in 2Fe-2S proteins. The cubic Fe4(S2-)4 clusters in 4Fe-4S proteins may be vitualised as a cobination of two Fe(S2-)2Fe clusters of 2F2-2S ferredoxins.Each Fe in these Fe4(S2-)4 cluster is tetrahedrally coordinated by three acid labile sulphide sulpher(S2-) and one cysteine sulpher (cys-S) as shown in the Fig.The oxidised formThe oxidised form contains three high spin Fe(III) and one high spin Fe(II) and its paramagnetism is equivalent to one unpaired electron due to antiferromagnetic interaction.

The oxidised form of HIPIP is e.p.r. active(g=2.02)

The reduced form contain two Fe(III) and two Fe(II) and is diamagnetic due to antiferromanetic interaction.These proteins, therfore, function as one-electron carriers:

8Fe-8S, FerredoxinsThes ferredoxine are small (MW=6000 daltons) and cosist of two 4Fe-4S, clusters situted at 12A0 apart each of which can undergo one electron change with E0 = -0.4volts.

As a result, the whole protein functions as atwo-electron carrier.The oxidised form contain equal number of Fe(III) and Fe(II), but shows lower magnetic moment(1.2B.M. per iron) due to extensive antiferromagnetic coupling and spin delocalisation