8 myoglobin and hemoglobin

what is the function of a protein determined by

the structure

what is the structure of myoglobin

•Monomer

• 1 subunit/1 polypeptide chain - 153 aa and 1 domain (bc less than 200)

• has only tertiary structure - doesnt have quaternary

• has 8 alpha helices and irregular structures (loops)

• has heme prosthetic group

• has hydrophobic pocket between helix E and F for heme since its hydrophobic but amphipathic

what is the structure of hemoglobin

•Oligomer - atleast more that 1 subunit

• 4 subunits - Each subunit has 1 domain

• also has quaternary structure - heterotetramer (atleast two are different, 2 alpha and 2 beta)

what is the main function of red blood cells

• has hemoglobin (doesnt have mitochondria) that binds to oxygens in the lungs and releases it in the tissues that need it

• O2 structure is not polar and not super soluble in water which is why you need help transporting it

what is the function of myoglobin

• Act as local reserve of O2 during intense exercise

• Store O2 in aquatic animals

• cytoplasmic protein found in muscle

• Facilitate O2 diffusion through muscle tissue - by allowing it to move quicker from plasma membrane to inner mitochondria membrane in aqueous solution

• Inactivating Nitric oxide and removes reactive O2 species because their unstable

what does the function of proteins also depends on

• depends on their ability to bind other small molecules (ligands) reversibly

• The greater the affinity of Protein X for ligand Y the more XY we will have at any concentration of Y or X - the interactions that link them is h bond, ionic interaction, hydrophobic, dipole-diople - the more you interact the better you can bind protein to ligand

what do ligand binding curves look like

• hyperbolic curve, as the concentration of the ligand increases a lot the binding plateaus because all binding sites are full

• as affinity increases the Kd lowers

what is Kd

measures for any [ligand] how much is and isnt bonded

how does the ligand binding curve change depending on the affinity

Y has the lowest Kd → highest affinity

Z has the highest Kd → lowest affinity

• lower affinity and higher Kd shifted to the right

Why does myoglobin need a prosthetic heme group

• to carry oxygen bc side chains cant and important for structure

what is the structure of heme

• circular and planar - doesnt have a protein

• In heme, the porphyrin ring contains an Fe2+ ion coordinated between the four N atoms - can form 6 coordination bonds which are covalent and 1 atom donates both electrons - these keep iron tightly bound

• has to be Fe 2+ to bind to oxygen

• Note that the two substituents at the bottom of the ring are polar propionyl groups whereas the rest are non-polar aliphatic groups.

• it is amphipathic

• see slide 11

which histine does heme form a coordination bond

• His F8 aka His 93 - proximal histidine which permanently attaches heme to globin

• on fifth coordinate position of Fe2+ ion

how is the porphyrin ring help in place

by hydrophobic interactions and by coordination bond between Fe2+ and a histidine (aa 93) His F8 which is proximal

where does oxygen bind to the heme group

• on 6th coordinate position on the Fe2+ ion - always binds at an angle

• the distal histidine or His E7 assists in oxygen binding by forming H bond with it - only interacts with oxygen not iron, doing this helps carbon monoxide not being able to bond

what is prevented when heme binds to polypeptide

Heme binding to the polypeptide helps prevent oxidation of Fe2+

what are two qualities that the oxygen binding sit in myoglobin exhibits and examples

Binding sites are designed precisely to optimize binding affinity and specificity

• when heme is not with globin - CO binds to heme with > 20,000 times the affinity of O2, CN- and H2S also bind here with high affinity

• when heme is attached to globin - Now CO binds to heme in myoglobin

  with only > 200 times the affinity of O2 - because oxygen likes to bond at an angle

• see slide 19

what curve does oxygen binding to myoglobin have

• hyperbolic curve that tells you binding is independent of other molecules

• lowest affinity when active muscle to offload oxygen, and resting muscle has plateau because you want myoglobin to load up with oxygen

how does pH effect myoglobin binding

small changes dont effect ability of myoglobin binding to oxygen but it does for hemoglobin

what is an apoprotein

• known as this with out prosthetic group

• when it has prosthetic group it is known as holoprotein

what subunits does the quaternary structure of hemoglobin have

Tetramer with two types of globin: its a heterotetramer

2 identical aplha subunits

2 identical beta subunits

• each subunit has a prosthetic group

• has 4 binding sites for oxygen (4 hemes interact with oxygen) but myoglobin only has 1 site

how many helices, pp chains, and location of hemoglobin and myoglobin

Hemoglobin (Hb) - found in RBC so its a cytosolic protein (doesnt have disulphide bridges)

• 4 polypeptide (pp) chains, 2 alpha chains, 2 beta chains

• Each pp chain has 8 alpha-helices, loops and 1 heme and one hydrophobic pocket, Hb binds 4O2

• In erythrocytes

Myoglobin (Mb)

• 1 pp chain with 8 alpha-helices, loops and 1 heme

• Mb binds 1O2

• In myocytes - cardiac and skeletal muscle cells

what are conservative substitutions

-these have minor effects on structure - not being a big change, changing one AA for another

• Leu to Ile, Thr to Ser (both have hydroxyl group and similar size

what are critical substitutions

can change structure and function- depending on location (His - Lys)

• Ser - Val

• when choosing you look for the biggest change

what do the tertiary structures of beta-globin, alpha-globin, and myoglobin have in common

• All 3 polypeptides comprise 8 alpha- helices with a heme binding pocket between helices E and F (hydrophobic)

  • alpha and β subunits are ~40% identical in 1o sequence

  • alpha subunit and MB are ~18% identical in 1o sequence

  • mainly conservative substitutions

• Homologous proteins - have similar secondary and tertiary structures but not similar primary structures

Predict the structure of neuroglobin, a monomeric protein

- 8 helices, no sheets, has E&F, hydrophobic pocket, heme prosthetic group

how does the alpha subunit and beta subunit of Hb bind to oxygen

bind O2 in exactly the same manner as myoglobin

⚫ Each binds oxygen at the 6th coordination position of an Fe2+ ion in a heme ring.

• Several critical residues in the oxygen binding sites are invariant (never change) among the three polypeptides - critical for function of proetin

  • his F8 - proximal

  • his E7 - distal

what is a hyperbolic curve indicative of

indicative of constant affinity generally high

• Ligand affinity (Kd) does not change - E.g. Myoglobin.

• typically, monomers

▪ One binding site for ligand that rarely changes

what is a sigmoidal curve a diagnostic of

diagnostic

of cooperative binding affinity.

• Ligand affinity changes as more ligand binds - E.g. Hemoglobin (4 binding sites)

• typically, oligomers - more than 1 subunit

▪ More than one binding site for the ligand

what type of binding does a sigmoidal curve represent

• oxygen binding to hemoglobin

• high affinity state in lungs to pick up oxygen

• in tissues ther hemoglobin goes into low affinity and offloads oxygen for resting muscles

• in active muscle the hemoglobin offloaded a lot of oxygen so myoglobin acts as a reserve for oxygen

• Cooperative process

• Necessary for efficient O2 delivery

• Reflects a change in binding affinity

**theres no myoglobin in lungs

• At 20-40 Torr Hb and MB must have different affinities for O2

what are the similarities and differences of the functions in Mb and Hb

• Similar functions

  • Reversibly bind/release O2 - myoglobin holds it tighter bc its a reserve

• Different functions

  • Mb: O2 transport/storage within tissue

  • Hb: O2 transport from lungs to tissues

what are the two distinct structure for hemoglobin

tense T state - low affinity, has large central cavity

relaxed R state - high affinity, small central cavity

• In deoxyhemoglobin, a His residue on the beta subunit fits between a Thr and a Pro residue in the alpha subunit - T state

• Upon oxygenation, the hemoglobin changes shape and the His residue is now located between two Thr residues on the alpha subunit - R state

  • conformational change in Hb structure

what is allostery

The binding of a ligand at one site on a protein affects the binding of ligands at other sites

• can be effectors that are homoallosteric or heteroallosteric

what are effectors

compounds which, upon binding, alter affinity at other binding sites

• can increase or decrease but you dont know

what is homoallosteric and heteroallosteric

• Homoallosteric – binding of the effector affects further binding of the same compound - Typically referring to ligands increasing its own affinity

  • Sigmoidal curve in presence of ligand

• Heteroallosteric – binding of the effector affects further binding of a different compound

  • Activators – increase binding affinity of ligand

  • Inhibitors – decrease binding affinity of ligand

**see slide 40-42

what are the events in oxygen binding to hemoglobin

• T-state (no O2 bound)

• O2 binds to a subunit

  • Fe2+ moves into plane of heme

    • Histidine F8 moves with iron

    • Helix F moves - gets pulled

    • Subunit interface changes

• Subunit interface change affects other subunits

  • Helix F/His F8/Fe2+ movement into plane of ring

  • Oxygen binding site becomes high-affinity (R)- Fe2+ moves into plane of heme rings

    • Oxygen binds more readily to these sites.

what bonds break when you shift from T to R

The shift from T to R breaks the salt bridges that hold BPG in place

• happens in lungs

• space in central cavity decreases

what are the 4 allosteric effectors for hemoglobin

• O2 - favours R state, positive effector/activator

• **BPG (2,3-bisphosphoglycerate) - favours T state, negative effector/inhibitor

• H+ - favours T state, low pH = negative effector/inhibitor

• CO2 - produced in large amounts during high cellular activity, acts indirectly** and directly to stabilize the T state, favour T state

what does BPG stabilize

BPG is essential in stabilization of the T state of Hb

• negative heteroallosteric effector (inhibitor) of oxygen binding

• has 5 negative charges

what does BPG bind to

• 1 BPG binds in the central cavity of deoxyhemoglobin (T state) (1 binding site for BPG and 4 for oxygen)

• The negative charges on BPG interact with positively charged groups on the protein that are directed into the central cavity.

• The central cavity in oxyhemoglobin (R state) is too small to accommodate BPG

• when you increase BPG the S curve shifts away from the y axis to the right

what is the indirect effect of CO2

H+ ions facilitate formation of the T state- enhances BPG binding

• The effect of [H+] (pH) on Hb’s O2 binding behaviour is called the Bohr effect - so more CO2 produced = more H+ produced = histidine protinated

• CO2 is not very soluble in aqueous blood so most gets taken back to lungs by converting it to HCO3-

• when pH decreases the histidine become protinated and forms a salt bridge with BPG

what is the bohr effect

• Metabolism generates protons (lowers pH; more [H+]):

  • ATP + H2O → ADP + Pi + H+

  • CO2 + H2O → HCO3- + H+

• Lowering pH leads to protonation of side chains:

  • His + H+ → His+

• His’s associated with BPG binding become protonated

  • Enhance BPG binding, Reduce O2 binding bc your stabilizing T state

• Subunit interface is affected

  • New electrostatic interactions form

how does capillaries and lungs use CO2

• lungs convert biocarbonate ion and H+ ions into water and carbon dioxide

  • histidine is deprotinated bc pH is more that pKa and wont interact with BPG so well

• capillaries in muscle take carbon dioxide and water into biocarbonate ions and H+ ions

  • histidine become protinated

how does the saturation curve change in the lungs and skeletal muscle

• pH of blood in lungs is 7.6 (above physiological pH of 7.4) so theres high pH and low hydrogen ion concentration - high affinity so it shifts to the left (closer to y-axis)

• pH of blood in skeletal muscle is 7.2 (below physiological pH) so gives low affinity for oxygen, high hydrogen concentration, low pH, histidine is protinated and helps stabilize T state

what are the 5 combined effects of oxygen, BPG, and pH on Hb function

• In the lungs and in the tissues, any given molecule of Hb can exist in either its T form or its R form.

• The proportion of molecules that are in either form (the position of the equilibrium) depends on the presence of CO2, 2,3-BPG, on the [H+] ions, and on the ppO2 (main driving force if youre R or T)

• The proportion of molecules that are in either form (high or low affinity) determines how much oxygen is bound or released.

• The lungs have a high pp O2 and a relatively high pH (low [H+]), The R state is thus favored, and when oxygen binds it triggers the switch to the R form.

• Actively respiring tissues have a relatively low pH (high [H+]), high levels of CO2 and a low ppO2, The T state is favored, and oxygen is released.

describe the pH, BPG binding, and O2 binding in lungs and skeletal muscle

In Lungs:

High pH → low BPG binding to HB → high O2 binding

In Skeletal Muscle:

Low pH → high BPG binding to HB → low O2 binding

what are two physiological disease/adaptation with AA substitution

• Amino acid substitutions may be disastrous or physiologically significant

  • Sickle cell anemia – a genetic disease

  • Fetal hemoglobin – a physiological adaption

what are sickle red blood cells

beta chain Glu6 is replaced with Val (critical bc youre going from charged to hydrophobic)

• RBC have to be mobile and squeeze through smaller capillaries so sickled ones cant

what is the effect of the sickle cell mutation

• In Hb there is a small hydrophobic surface patch which is exposed between the E and F helices during the transition from R to T form.

• The hydrophobic Val binds here, causing the Hb molecules to aggregate into long polymers/fibres - which becomes rigid and sickle shape and not mobile enough, recessive disease, found in areas with high malaria

what is fetal hemoglobin

• 2 alpha and 2 gamma subunits - can be a treatment of sickle cell

• gamma-subunit homologous to the adult beta-subunit.

• substitution of His143 for a Ser.

  • This is one of the His residues that is involved in binding BPG.

• In its absence, BPG binds with a lower affinity, and so the T state is less stable at any given ppO2 and [H+] ions.

  • In pregnancy, there is a 30% increase in 2,3-BPG

HbF (fetal) vs HbA (adults) affinity for oxygen

HbF has a higher

affinity for O2

than HbA

what are 3 specific roles for His residues in Hb function

• His F8 aka proximal

  • Attachment of heme as prosthetic group to globin

• His E7 aka distal

  • Assist O2 binding by forming h bond with it

  • Decreases affinity of CO

  • Geometry of substrate binding site

• 4 His in central cavity

  • BPG binding