Bio Inorganic Ferritin, Hemoglobin, and Hemerythrin PDF

 Ferritin Ferritin is a blood protein that contains iron. A ferritin test helps your doctor understand how much iron your body stores. Ferritin is a universal intracellular protein that stores iron and releases it in a contro...

 Ferritin Ferritin is a blood protein that contains iron. A ferritin test helps your doctor understand how much iron your body stores. Ferritin is a universal intracellular protein that stores iron and releases it in a controlled fashion. The protein is produced by almost all living organisms, including archaea, bacteria, algae, higher plants, and animals. In humans, it acts as a buffer against iron deficiency and iron overload. Ferritin is a globular protein complex consisting of 24 protein subunits forming a nanocage with multiple metal–protein interactions. It is the primary intracellular iron-storage protein in both prokaryotes and eukaryotes, keeping iron in a soluble and non-toxic form. Ferritin that is not combined with iron is called apoferritin.  Apoferritin Ferritin Ferritin Red-brown colour - Aspherical shaped molecule (hallow sphere) – Apo ferritin - Filled with iron – ferritin - Iron can be stored within the cells in a non-toxic form. - High concentration in liver, spleen, and bone marrow - Ferritin contains a shell of protein surrounding a core that contains the iron CORE: (formed via a process - Biomineralization) Iron crystallizes together with phosphate & hydroxide ions Diameter ~ 40- 80 Å (up to 4500 Fe atoms) Quasi crystalline core (gives XRD/ED pattern) Closed-packed array of oxide ions and OH- ions with Fe atom in Composition approximately close to: the Oh interstices. 8(FeOOH). FeO. H2PO4 Phosphate is not a art of bulk structure but play some role in covering (Ferric oxyhydroy phosphate complex) (FeOOH)x particles or attaching (FeOOH)x particles to each other and to the protein sheath. XRD Pattern similar to 5Fe2O3.9H2O (Ferri hydrate) – No phosphate Slow addition of NH4OH “FeOOH” – spheres up to 7000 pm in diameter Fe(NO3)3 Hydrolysis in slightly basic (Ferric Nitrate) solution HCO3-  Magnetic Behaviour – Mössbauer Spectroscopy Fe3+ High-Spin State & Anti ferromagnetic coupling S = 5/2 PROTEIN: The protein core around the ferritin – apo ferritin 24 subunits, Mol. Wt= 480 kDa apo ferritin Allows controlled access to the core through 8 hydrophilic channels (3-fold axis) and core 6 hydrophobic channels (4-fold axis) 12 nm These channels have different chemical composition (Transmission Electron Microscope, TEM images) Fe core density is 2.5x that of apoferritin Volume of iron core is ¼ of the whole. It appears that Fe3+ enters via hydrophilic channels and leaves via hydrophobic channels. Average diameter of the channels ~ 0.4 nm (X-ray) Molecules larger than 0.4 nm (0.7-1.0 nm) can also penetrate into the interior due to conformational flexibility - “breathing”  The 4-fold channel, which is lined with leucine residues, is hydrophobic ("water-hating," lined with nonpolar side chains). The 3-fold channel, which is lined with glutamate and aspartate residues, is hydrophilic ("water-loving," lined with polar side chains). The 3-fold channels are lined with the polar side chains aspartate and glutamate and make the channel hydrophilic.2 The hydrophilicity of the channel allows for the transport of water, metal cations, and hydrophilic molecules of an appropriate size into and and out of the ferritin center.4 Most studies indicate that the 3-fold channel is the main channel for Fe2+ ions both into and out of the cell.1,3 The 4-fold channels are lined with the non-polar side chain leucine and make the channel hydrophobic. It is widely thought that the 4-fold channels are involved with the diffusion of oxygen and hydrogen peroxide into and out of the ferritin center.1 1] Bou-Abdallah, F. The Iron Redox and Hydrolysis Chemistry of the Ferritins. Biochimica et Biophysica Acta (BBA) - General Subjects 2010, 1800 (8), 719–731. Crabb, E.; Moore, E. Metals and life; Royal Society of chemistry: Cambridge, 2010. Theil, E. C.; Tosha, T.; Behera, R. K. Solving Biology’s Iron Chemistry Problem with Ferritin Protein Nanocages. Accounts of Chemical Research 2016, 49 (5), 784–791. FUNCTION: e- Iron storage in a non-toxic form and transport when it required. Fe3+ Fe2+ Dithionite OR Ferritin Fe core Apo ferritin (Red brown colour) Thioglycollate (Colorless) (reduction) pH= 4.6-5.1 How Fe can be released? To be released it has to be dissolved first! i.e. breakdown of Fe lattice Reacts with water to form “water cage” e- around Fe2+, thus forming a hydrated Fe2+ It is accomplished by Dihydroxy fumarate Fe3+ Fe2+ ion. Released from ferritin via protein 3-fold channels due to polarity. 3-fold channels While going thorough the channel it probably not as Fe(H2O)62+ - since it cannot fit through the channel  Transferrin - Tf (Fe collector) carbohydrate + protein Transferrins are glycoproteins found in vertebrates which bind to and consequently mediate the transport of Iron (Fe3+) through blood plasma. It is produced in the liver and contains binding sites for two Fe3+ atoms. Distorted Oh Transferrin has a molecular weight of around 80 kDa and contains two specific high-affinity Fe(III) binding sites. Transferrin glycoproteins bind iron tightly, but reversibly. Tf-receptor Bioinorganic Chemistry, Dieter Rehder (Oxford) The affinity of transferrin for Fe(III) is extremely high (association Tf-Fe Two-phenolate oxygen One imidazole nitrogen constant is 1020 M−1 at pH 7.4) One carboxylate oxygen Two carbonate oxygen Liver Role of Fe-Tf: Cell membrane secretion 1. Fe is now soluble under physiological condition Ferritin Transferritin 2. Iron mediated free radical toxicity is absent (storage) (Transport) 3. Facilitate Fe transport into cells (in plasma) Mechanism of Fe loading and unloading is not known. Binding constant ~ 1026 [Efficient scavenger of Iron]  Hemoglobin -Function – Dioxygen (O2) transport -An approximate tetramer of myoglobin (Mb) -Mol. Wt 64500 g/mol -4 heme groups bound to 4 protein chains 4 “quaternary” globin protein subunits The quaternary structure of a protein is the association of several protein chains or subunits into a closely packed arrangement. Each of the subunits has its own primary, secondary, and tertiary structure. The subunits are held together by hydrogen bonds and van der Waals forces between nonpolar side chains. 2 (146 amino acid), 2 (141 amino acids) chains 4 Peptide chains are bound by H bonding, salt bridge (bonds between oppositely charged residues) and hydrophobic interaction Heme (PROTOPORPHYRIN IX (PIX)) T- State R- State Other Natural O2 carriers: In lower animals the respiratory metallo-proteins differ from Hb. Most important two are: Hemerythrins (Hr) and Hemocyanins (Hc)  Hemerythrin CAREFUL: Hemerythrin does not, as the name might suggest, contain a heme. Function: O2 transport and storage 4-fold symmetry The essential structural units – single chain protein 13-14 kDa 115 amino acids residues In BLOOD: Octomer – tertiary structure Contains 2 Fe atoms Fe atoms bound by 2-carboxlic (Aspartic/glutamic acids), 5-imidazole nitrogen (Histidines) Trimeric Single Oxygenated Hemerythrin Protein Complex Hemerythrin protein In TISSUES: monomers, dimers, trimers or tetramers Each O2 binding sites contain only two Fe(II) atoms  Active site of hemerythrin before and after oxygenation. -OH H Bond Hydro Peroxo group One vacant site II II III III Resonance Raman (o-o) = 844 cm-1 Colorless -Oxo Typical value for DEOXY OXY Singly bound peroxides Purple (480 nm) High-Spin dFe-Fe ~ 3.57 Å dFe-Fe ~ 3.24 Å Exchange Coupling energy, J = -100 cm-1 Exchange Coupling energy, J = -10 cm-1 Antiferromagnetic interaction (Magnetic susceptibility) Two non-equivalent Fe sites (Mossbauer spectroscopy) Mossbauer spectroscopy = Fe2+ (H.S) Hemoerythrin (O2) : 844 cm-1 “Peroxo” type (O2) : 798 cm-1 “Peroxo” type (Fe-O2) : 500 cm-1 (Fe-O2) : 478 cm-1 Single oxygenated functional unit from the Electron microscopical images of hemolymph proteins of various springtails. The “single hemocyanin of an octopus droplet negative staining“ technique and 2% (v/v) aqueous uranyl formate was applied for the preparation of the sample. (a) Overview of the hemolymph proteins of Orchesella villosa. A huge amount of the 2 × 6-meric protein is present in the hemolymph. Inserts show 2 × 6-meric proteins found in the hemolymph from Isotoma viridis (b), Folsomia candida (c), Coecobrya tenebricosa (d), Orchesella cincta (e), and Sinella curviseta (f). (g) Superposition of 13 images of Sinella curviseta hemocyanin for gaining a better contrast. Biomolecules 2019, 9(9), 396; https://doi.org/10.3390/biom9090396 Oxy-Hc extracted from Cancer magister (Pacific crab) and Busycon canaliculatum (channeled whelk) exhibit absorption bands near 570 and 490 nm. Pacific crab channeled whelk https://vimeo.com/311696267  Ferredoxin (Fd) Non-heme Fe-S proteins “Biological Capacitors” - Can accept or discharge electrons by Function: mediates electron transfer in a range of metabolic reaction changing the ox.st of Fe (+2, +3)  Labile S or Inorg. S Triple bridging S (sulfide ions) (Non-Labile S: sulfhydryl group) Td 2Fe-2S iron-sulfur cluster binding domain Cubane D2d 4Fe-4S iron-sulfur cluster binding domain Cysteine = (HSCH2NH2CHCOOH) Fe2(2-S)2 Fe4(3-S)4 Two types of S. The labile S can be easily removed by washing with acids (RS)4Fe4S4 + 8 H+ (RS)4Fe48+ + 4H2O sulfhydryl group Labile S  Distorted Td, e- transfer at low potential ! Fe1S0 The test differentiate Fd from Rubredoxins (no inorganic S) Single Fe site (Fe2+/Fe3+) H.S. State Rubredoxin 2Fe2S Magnetism: Oxidized - [2FeIII-2S]2+ Magnetism: Reduced - [2FeII&III-2S]+ 2 H.S state Fe3+ ions bridged by S2- dimer Two non-equivalent Fen+ sites And each Fe3+ coordinated by two labile S atoms One electron reduction – Total number of d electron = 11 (odd) Diamagnetic @ very low Temp! (EPR silent!) Anti ferromagnetic coupling persist. The added e- behaves as doublet. Paramagnetic S= ½ (EPR signal) 278 pm S=5/2 S=5/2 S=2 S=5/2 (super exchange through -S- bridge) Net S=0 Net S=1/2 Structures of biological Fe/S clusters: a) Rubredoxin, b) [2Fe2S]-ferredoxin, c) Rieske-center, d) [4Fe4S]ferredoxin, and e) [3Fe4S] cluster.  4Fe4S Low potential iron protein High potential iron protein (HiPIP) The formal ox st can be +3, +2 or +1, but in any given biological system only one pair is employed. S= ½ S= 0 S= ½ Fe2.75+ Fe2.5+ Fe2.25+ Fe2+ CORE OX ST [Fe2+ + 3Fe3+] [2Fe2+ + 2Fe3+] [3Fe2+ + Fe3+] [4Fe2+] HiPIP Couple Fd Couple Cubane type model compound  Blue Copper Proteins Plastocyanin (Pc) - A protein containing single Cu atom - found in higher plans, algae, blue green algae Role: Photosynthesis  Pc-CuI Pc-CuII + e- Plastocyanin was the first ‘blue’ or ‘type 1’ copper protein 1. Intense blue color (Abs. ~ 600 nm) 2. Low hyperfine coupling (hfc) in EPR in in gII region 3. High redox potential CUPREDOXINS strong Cys–Cu2+ charge transfer band Neither Td nor SP This intermediate (flattened Td) geometry facilitates e- transfer.  Cytochromes (Cyt) Cytochromes are redox-active proteins containing a heme, with a central Fe atom at its core, as a cofactor. They are involved in electron transport chain and redox catalysis. They are classified according to the type of heme and its mode of binding. Four varieties are recognized by the International Union of Biochemistry and Molecular Biology (IUBMB), cytochromes a, cytochromes b, cytochromes c and cytochrome d. Cytochrome function is linked to the reversible redox change from Fe(II) to Fe(III) oxidation state of the iron found in the heme core. In addition to the classification by the IUBMB into four cytochrome classes, several additional classifications such as cytochrome o and cytochrome P450 can be found in biochemical literature. Various functional groups attached to Cyt tune the delocalized MO of the complexes and thus vary its redox potential.  In porphyrin the M-N distance is ~200 pm long. The size of the “hole” in the center of the ring is ideal for accommodating first row transition elements. Porphyrin ring is rigid – due to delocalization of pi electron in the pyrrole ring The Ox state during operation - Fe(II) or Fe(III)  The reduction/Ox. Potential is such that the electron flow is b c a O2 S S Cyt c: The heme group is attached to a polypeptide chain and wrapped around it. The number of amino acids vary from 103, 104 to 112. Iron 5th and 6th coordination sites have been occupied by histidine N and S from methionine segment. All coordination sites have been occupied (unlike Hb and Mb) Electron Transfer Process: Reduction of O2 to H2O  The reduction/Ox. Potential is such that the electron flow is b c a O2 Cytochrome c Oxidase Extended X-ray absorption fine structure (EXAFS) Cytochrome c Oxidase reduces dioxygen (O2) to water. This requires four electrons and four protons. Electrons are donated from the electron carrier cytochrome c and the four protons are transferred from the matrix via several pathways. Each cytochrome c only carries one electron, thus four cytochrome c molecules must be reduced to complete the reaction. In the process of dioxygen reduction, CcO also pumps four protons across the inner membrane. The net reaction is as follows: 4Cyt cred + 4H+ + O2 + 4H+matrix → 4Cyt cox + 2H2O + 4H+intramembrane space  O2 H 2O B Cyt a3 J. Biol. Chem. 1977 252: 776-785.  Chlorophyll (Chl) Photo-induced electron transfer function (Little Known!) Green Pigment - present in the thylakoid membrane of the chloroplast  Chl-a Chl-b I 2 3 II 1 4 8 5 IV 7 10 9 6 III Phytyl group Phytyl group (23 C long)  -porphyrin family (Chlorin) Chl-a -not a heam I 2 3 II -Mg2+ center located 0.3-0.5 Å above the non-planar macrocycle plane 1 4 -A fused cyclopentanone ring group between III and IV, connected 6th C, exhibits keto-enol equilibrium, strongly favours keto-form -The long phtyl group serves to bind Chl to cell membranes (in the cell) -One pyrrole is reduced to C7-C8 8 5 -Extensive conjugation of porphyrin ring, decreases the electronic transition energy IV 7 10 9 6 III and shifts the absorbance maximum into the visible region (~600-700 nm) Optical Spectra Phytyl group (23 C long) Photosynthesis: + + 4e- [Water splitting] + [Reduction of CO2] Here, both Cyts and Fds are involved in the e- transfer process, that is initiated by oxidation of photo-excited Chl. TWO REACTIONS CENTERS (P680 & P700) Energy of light becomes converted into chemical energy They absorb different energy photons 680 and 700 nm They differ only in the type of Chl present & accessory chemicals for the processing of trapped photons Either one of them initiates redox reaction - - - - - = 700 nm = 680 nm ATP Calvin Cycle Produces G3P (glyceraldehyde triphosphate) [o] 4e - [Reduction of CO2] [Water splitting] Mn4 Butterfly Clusters - - Mn nuclear spin : 5/2 3 3 3 2 3 The concentration of Chl at the active site is very small. Therefore, Caroteins undergo photoexcitation and transfer the photoexcited energy to Chl. Chl dimerizes in solvents that poorly coordinate with Mg2+. The keto-group at C-9 of ring V coordinate to Mg2+ of a second Chl through the carbonyl oxygen.  Synthetic Leaf Red Light (740 nm) Potential difference 422 mV and 24 A current  Enzymes Enzymes are catalysts in biological systems, they are built by proteins Controls rate of reaction by favoring certain geometries in the transition state Decreases the activation energy to form product. The molecules upon which enzymes may act are called substrates, and the enzyme converts the substrates into different molecules known as products. The enzyme is not destroyed during the reaction and is used over and over. uncatalysed https://www.genome.gov/genetics-glossary/Enzyme  Carboxypeptidase A (CPA-1) Carboxypeptidase A usually refers to the pancreatic enzyme that hydrolyzes peptide bonds of (carboxy) C-terminal residues with aromatic or aliphatic aminoacid side-chains. CPA-1 Preferentially cleave substrates with aromatic side chains  Td CPA contains protein chain of 307 amino acids and 1 Zn(II) ion Mol Wt: 34600 Egg-shaped molecule Max. dimension 5000 pm; min dimension 3800 pm. Active site is Zn(II) situated near the protein surface. d10 - No electron transfer. Lewis acid (accept an electron pair) 2N (His-69,169), 1O (Glu-72) 4th coordination site is free to accept electron from a donor atom in the substrate to be cleaved. The active site contains a hydrophobic pocket, that promotes the binding of aromatic side chains. The carbonyl group of the amide linkage coordinates with Zn for action. X-ray  (Lock and key mechanism) Tyrosine phenolic OH group moves 1200 pm close to imido group to form H bond with substrate. tyrosine Base catalyst – glutamate removal of water proton These two interactions are not only hold the substrate to enzyme but helps to break N-C bond (b) Probable interaction of polarized Water in the breaking of the amide linkage Arginine Arginine chain moves to 200 pm (a) Positioning of the substrate on the enzyme: to COO- of substrate. (i) C=O Zn; coordinate covalent bond (ii) H bonds: Arginine to carboxylate Tyrosine to amide (iii) van der Waals attraction: Hydrophobic pocket to aromatic ring (iv) Dipole attraction: glutamate carboxylate oxygen to carbonyl group (nucleophilic attack!) (c) Removal of products  Spectroscopy: Zn(II) is replaced by Co2+ (d-d transition; retains the enzyme activity). CPA (Co2+) spectrum – (absorptivity) molar extinction coefficient,  Irregular “Td” (probably necessary to effect the reaction) ENTACTIC – form of a metal : Lowering of the TS energy (by favoring some geometry) of the metals in the enzyme.  Carbonic anhydrase Carbonic anhydrase is an enzyme that assists rapid inter-conversion of carbon dioxide and water into carbonic acid (protons and bicarbonate ions). It has been found to be abundant in all mammalian tissues, plants, algae and bacteria. M.W: 30 K Active site Zn is coordinated to 3 histidine (94,96,119) and water or OH- Zn lies in the deep pocket The enzyme enhances rate of hydration in either direction by 106 times or more. Uncatlayzed: k = 10-11 S-1 Catlayzed: k = 104-106 S-1 The relative bonding power of Zn ion is reversed in the enzyme (I->Br->Cl->F-) due to softening effect of the apoenzyme on Zn. An apoenzyme is an inactive enzyme, activation of the enzyme occurs upon binding of an organic or inorganic cofactor I-