Hemoglobin

hemoglobin is protein that carries oxygen in blood to transport it

• oxygen is NP and insoluble in aqueous solution

hemoglobin is in red blood cells when blood is oxygenated, pressure of O2 high in lungs hemoglobin grabs hold of the blood and blood travels to capillaries and hemoglobin drops of O2

• process repeats

hemoglobin is heterotetramer with quaternary structure

• meaning 4 subunits that are not identical

  • 2 alpha subunits

    • 7 helices A-H but missing D

  • 2 beta subunits

    • 8 helices A-H

  • each subunit has a heme (prostatic group non protein that binds O2)

    • has 4 hemes so bind O2 in 4 places

  • suitable for O2 transport

myoglobin: 8 helices A-H

• 1 heme and only tertiary structure

• suitable for O2 storage

• stored in muscles and has a strong affinity for O2

  • useful when concentration of oxygen drops in blood like during anaerobic respiration myoglobin releases it then it diffuses through muscle cells and mitochondria pick it up for ATP sythensis

• higher concentration of myoglobin in deep see mammals, the longer the animal can go without O2

binding affintiy between Protein & Ligand

• enzyme and substrate type situtation but with protein and ligand

• Ligand is a small molecule that binds to binding site of protein, is suited for its protein

  • needs to have charge, shape, size, hydrophobicity must complement protein

• by plotting fraction of protein bound to ligand and ligand concetration increases can see how well a protein binds to ligand

  • as start adding ligand the staured the solution becomes giving more chance to bind to protein

    • at start this is rapid but curve starts to slow as binding sites decreases

    • when concentration is high enough 100% of protein will be bound to ligand

P50: point at which ½ of protein is bound to ligand

• x-axis is in terms of pressure for O2

• for hemoglobin it is 28 torr

• for myoglobin it is 3 torr

hyperbolic curve: one ligand binding site or multiple negatively cooperative binding sites: myoglobin

• binding sites work indenpendtly of each other

sigmoidal curve: multiple binding sites and cooperative: hemoglobin

• steep slope means high affinity for ligand

why is hemoglobins sigmoidal curve important

• has to regulary bind and drop of O2, needs the high and low affinity to do this

cooperativity: binding of 1 ligand affects affinity of remaining sites, initially difficult to bind ligand but once one is bound then more and more can bind

2 conformations of hemoglobin

• Tense (T-state): deoxyhemoglobin, low O2 binding affinity

  • favored during low Pressure of O2

  • in tissues

• Relaxed (R-state): oxyhemoglobin, high O2 binding affinity

  • favored during high Pressure of O2

  • in lungs

multiple conformations means its tertiary/quaternary structure have multiple ways of being arranged in 3-D form

• for hemoglobin binding of one O2 at one subunit increases affinity of O2 for the other subunits

hemoglobin is most stable in T or R state

heme: oxygen binding site that is prosthetic group, non-proteinaceous molecule, found in each monomer added to protein during or after translation, uses iron to bind O2

• iron is found in the middle of the porphyrin ring structure

  • has coordinate covalent bonds, this bond is a type of covalent bond where 1 atom donates both electrons, occur between metal ions and ligands

    • Fe and 4 nitrogens

    • iron 2+ state binds oxygen reversible and iron 3+ can’t

Fe2+ in heme participates in 6 different bonds when oxygenated

• has octahedral geometry

• 4 bonds with Nitrogen

• 1 with histidine

• 1 with O2 at an angle places it next to another histidine residue called distal His, His E7 or His 64

iron has coodiranate bond with side of histidine residue called proximal histidine or His 93 or His F8

• His93 is the proximal histidine in myoglobin chain

  • for alpha chain it is His 87

  • for beta chain it is His 92

proximal and distal naming for histidine in relationship to heme

nitrogen in distal His acts as a H bond donor for the oxygen which is the H bond acceptor

• diatomic oxygen can’t be a H bond acceptor but is in this case because of the polar iron and oxygen bond

quaternary change happens when O2 binds to heme or is released from it

• heme is oxygenated has planar conformation with iron in middle of it

• heme is deoxygenated iron is repelled from porphyrin ring in direction towards proximal His, domed shape results

  • relays a change to position of F alpha helix

alpha 1 and beta 1 & alpha 2 and and beta 2 subunits are assoicated tightly due to hydrophobic effect forming dimers, held together by more than 30 residues

• dimer is chemical structure that is formed by linking of 2 similar sub units

• alpha 1 and beta 2 & alpha 2 and beta 1 are held together by 19 residues

• interactions keeping dimers together are stronger

if hemoglobin was treated with urea (denaturing agent) it would separate into its dimers

upon oxygenation the distance between the beta subunits grows more narrow

factors that stabilize R and T states

• low O2 T state favored; domed shape of heme

  • stabilized by larges # of ion pairs relative to R state at alpha 1& beta 2 and alpha 2&beta 1 interfaces

  • hemoglobin likes to arrange polypeptide in a particular way when no ligand bound

• high O2 R state favored; planar shape of heme

  • ion pairs that stabilize T state are broken

proximal histidine is key for cooperatively as it shifts F-helix upon O2 binding

• replacing proximal histidine with glycine the position of F helix was unaffected, no cooperativity and increase in O2 affinity (experiment done by Barrick and team)

different IMF take place in R and T state

• T state has greater # or ion pairs

The proximal histidine forms a covalent bond with heme iron, while the distal histidine forms a hydrogen bond with O2

negative allosteric effectors of hemoglobin, H+, CO2, and BPG

there is something in blood that decreases hemoglobin’s affinity for O2

• pure hemoglobin has higher affinity for O2

allostery: binding of ligand at one site affects binding of ligand at another

• positive stabilizes ligand-binding conformation

  • O2 for hemoglobin as it stabilizes the ligand binding conformation and is homotrophic same as effector (ligand)

• negative destabilizes ligand binding conformation

  • destabilize R-state

  • CO2, H+, BPG found in blood

    • negative heterotrophic effectors as they are different then ligands

CO2 one of the byproducts of cellular resipration, 3 ways CO2 transported back (get rid off

• 7-10% dissolves in plasma of blood

• 70% dissolves in blood as HCO3-

  • generates negative allosteric effectors protons

• 20% carried away by hemoglobin, called carbaminohemoglobin (second negative allosteric effector)

protons

• red blood cells have carbonate anhydrase which catalyses

the protons generated from this reaction, is transported by hemoglobin about 20%

when pressure of O2 high in lungs hemoglobin favors R state, and get O2

• equilibrium favors R state or oxygenated form of hemoglobin

• p50 of hemoglobin is lower

when pressure of O2 low in tissues T state favored for hemoglobin

• when the hydration of CO2 happens in tissues, the pressure of CO2 increases, increasing protons, decreasing pH, causes pronated form of hemoglobin to increase, making it give up O2

• protons don’t bond in the same place as O2 but in different residues

• p50 of hemoglobin is higher

increase in protons decreases hemoglobins affinity for O2, so hemoglobins p50 rises

Bohr effect: effects of pH on O2 binding curve

• as pH decreases p50 increases

salt bridge between R of histidine 146 and R group of aspartic acid 94, stabilizes T state

• happens only when protonated

• happens when pH decreases like in T state in tissues

myoglobin doesn’t display Bohr effect, isn’t effected by pH, while hemoglobin is

the histidine side chain pKa increases upon O2 release

• its R group pKa is 6, but it can change based on environment changes called pKa perturbation

• holds on to proton more when closer to aspartic acid

carbaminohemoglobin

• deoxygentated form of hemoglobin

• CO2 reacts with N terminin of four globin subunits to form a carbamate end which carries a neagtive charge

  • carbamate ends can participate in salt bridges that stabilize T-state

• release of protons also contribute to Bohr effect

2,3 BPG

• binds to central cavity of hemoglobin (this changes with T and R state, larger in T state)

• has a negative 5 charge so interacts with positive charges, so binds to conjugate acid forms of basic side chains and N terminal amino groups

  • lysine, histidine, and N terminus in central cavity

• 8 positively charge groups in B subunits positioned in central cavity that stablize BPG

• allows significant release of O2 (case for all the negative allostrotic effectors

  

fetal hemoglobin and BPG

• still has alpha 1 and 2 but instead of beta it has gamma 1 and 2

• when fetus inside mom the p50 is lower then adult hemoglobin

  • needs the stronger binding affinity to O2 to be able to steal it from the adult hemoglobin

  • not very good at dropping but doesn’t need to be because fetus is smaller then adult

  • very good at picking up O2

• has serine at 143 residue instead of histidine in beta subunit

  • and 6 positive charges to stabilize it so doesn’t bind as tightly