Oxygen Binding Flashcards
What are the main properties of interest for protein structure
- size, shapre, charge, hydrophobicity/philicity
*note even though a protein is over all neutral it can still have charged patches, same goes for hydrophobicity
What is meant by protein interactions are stable or transient
- ligangs can bind and unbind changing protein conformation, then change back (transient)
- ligands can also interact and be stable, the protein will not go back to original conformation
**protein function is determined by structure, binding changes tructure chanigng function
**reversible is the most dominant form of modification
What is a prosthetic group
- involved in stable interactions
- molecule that is permanently associated with a protein and required for its function
what is a ligand
- invovled in transient interactions
- molecule that is bound reversible by a protein
What is a bidning site
- region that interacts w/ the ligand
- complementary to ligand in terms of size, shape, charge, hydrophobic/philic proterties
- interactions are highly specific
- proteins can have more than 1 bidning site
what is induced fit
- structural adaptation between protein and ligand
- conformational change can make a binding site more complementary bc proteins are dynamic and flexible
what are the components of an enyme
Substrate (ligand)
- molecule changed by an enzyme
Catalytic site or Active site (ligand binding site)
- binds substrate and facilitates its chemical transformation
why do non enzymes require regulation
- binding protein: control affinity
- protein mediated transport: modulate transport function
*not having enzyme activity does not mean it does not have function
what are some characteristics of oxygen
- poorly soluble in aqueous solutions
- inefficient diffusion thorugh tissues
- larger multicellular organisms require mechanisms for transporting O2
- interaction of transporter with O2 MUST BE SPECIFIC AND REVERSIBLE
how is oxygen transported?
- complexed w/ transition metals with high affinity for O2 (ex: iron and copper)
- Free iron can promote the formation of highly reactive O2 species
- tendency is reduced when iron is incorporated into heme
What is heme
prosthetic groups
*prosthetic group= permanent modification, heme cannot come of but oxygen can
- Fe 2+ binds O2 reversibly
- Fe3+ does not bind O2
- in heme containing proteins ireversible oxidation of Fe2+ by O2 is prevented
- heme is buried within the protein
- one coordination bond is occupied by a side chain N of a His residue, O2 binds reversibly at remaining position
Heme = protoporphyrin IX + Fe2+
What are porphyrins
- heme is an example
- 4 pyrrole rings connected by methine bridges (-CH=)
- linked into a conjugated C=C double bond system
- the substitutions at the X define the type of porphyrin (in heme two V’s are propionate groups)
- the 4 N atoms can bind to a metal ion in the centre
*all have this common middle structure
what is Myoglobin, Hemoglobin and leghemoglobin
*oxygen bidning proteins w/ prosthetic group
Myoglobin:
- monomer, binds and stores O2 in muscle
Hemoglobin
- tetramer: 2 a-globins and 2 b=globins
- O2 transporter
Leghemoglobin
- found in leguminous plants
- sequesters O2, protecting O2 sensitive enzymes in N2 fixing bacteria
How are Globin structures named
- Globins are globular α helical proteins
- their 8 α helices are denoted: A-H (N-C terminus)
- connecting loops are identified by the two helicies they join (CD, DE etc)
- amino acids are identified by their relative position within that motif
*monomer, single polypeptide chain
- F8= the 8th aminon acid in helix F (in myoglobin is His93 and His87 in α-hemoglobin)
exaplin myolgobins heme binding pocket
- heme bidning pocked is formed by E anf F helices
- propionate side chains of heme are near the surface of globin
- the rest of heme is surrounded by non polar residues
***except 2 histidies residues which are polar***
what is the role of histidines
- one His F8 is directly bonded to Fe2+ (5th coordination bond) called the proximal histidine (F8)
- E7 His is close, but not bonded to heme (on the globin protein) distal histidine (E7)
- O2 binds to Fe2+ on E7 side of the atom (6th coordination bond)
**recall iron is molecule in the centre, linked by nitrogens, iron interacts with globin via histidine
**these His must always be in the same location, prox and distal his must be conserved in both myoglobin and hemoglobin
what is the proximal histidine
- His residue at F8 is directly bonded to Fe2+
- His F8 forms a 5th coordinatino bond for Fe2+
*look at text for other explanation
what is the distal histidine
- His E7 is close but non bonded to heme
- O2 binds to Fe2+ on the E7 side, O2 forms the 6th coordinatino bond
- distal histidine is positioned to interact with the O2 molecule
WHat is significant about histidine in myoglobin and hemoglobin
- Alignment of the sequences of myoglobin and both hemoglobin chains is shown
- The proximal and distal histidine residues are conserved in all three
*these His are alywas in the same sp
What is Mb
- simple O2 bidning protein (myoglobin)
- binding of O2 depends on structure of ligand binding site and flexability of protein
- this protein vibrates (called rbeathing): very tiny movements of amino acid side chains on nanosec time scale
*inside of globin is hydrophobic, the vibration allows oxygen to vibrate into small cavities to push its way in until it encounters the iron
how is O2 binding by globins measured
- conjugated double bond system causes strong absorption of visible light
- O2 binding affects electron distribution and alters abs of light by heme
- oxy & deoxy forms of heme have different absorption spectra (oxyheme abs more blue looking red and deoxy abs more red)
*this is why arterial (oxygenated blood) is red and venous (deoxygenated) is blue
- affects light abs of globins also so can be used to experimentally meausre
What is [P] [L] [PL] Kd and Ka
what is the equation for modeling reversible protein ligand interactions
what is the equation of occupancy (θ)
θ = binding sites occupied/total binding sites
θ = [PL] / [PL] + [P]
**** θ = [L] / [L] + Kd
*When [L] = Kd half of the ligand bonding sites are occupied
* Kd is equiv to molar conc of ligand at which half available ligand binding sites are occupied
*the more tightly a protein is binds to a ligand, the lower the conc of lig required for half the binding sites to be occupied
*myoglobin and hemoglobin differ in how tightly they bind their ligand
what is θ = pO2 / (pO2 + P50)
used for O2 bidning proteins
- pO2 = partial pressure of O2
- θ = occupancy
- P50 = partial pressue of O2 at which half the ligand binding sites are occupied
- look at graph, protein has extreamly high affinity for oxygen, can bind readily at low pressure
exaplain the structural basis of Hb/Mb specificity
- CO binds heme upright
- O2 binds heme at an angle
- Mb and Hb can increase selectivity vs heme because the protein
1) created steric hindrance between the distal His E7 and CO
2) make favourable hydrogen bond between O2 and His E7
exaplin O2 binding of myoglobin
- Mb binds O2 with high affinity
- wide range of Mb is insensitive to pO2
- pO2 in tissues is 4kPA
*Mb is essentially saturated
compare structure of globins
*tertiary and primary structure of hemoglobin and myoglobin are similar
compare globin sequences
- in diff globins, segments of (mostly) different amino acids form similar structures
- out of 150 amino acids only 27 (18%) are identical between Mb and Hb subunits
where are the interfaces in hemoglobin and how are subunits held together
- The strongest interactions between the subunits occur at the α1- β1 & α2 - β2 interfaces
- the type of interactions hold the subunits of hemoglobin together are
- hydrophobic interactions
- hydrogen bonds
- ion pairs (salt bridges)
*30 residues are involved in the interface between α1- β1 and between α2 - β2
*19 residues are involved in the interfaces between α2 - β1 and between α1 - β2
what is the role of ion pairs
- salt bridges
- stabilize the conformation of hemoglobin
- several important ion pairs occur at the α2β1 and α1β2 interfaces.
ex:
β1 subunit His HC3 forms salt bridges within the β1
subunit at Asp FG1 and with the α2 subunit at Lys C5
what is the T to R state transition
- Hb can undergo conformational change from a low affintiy state (T) to a high affinity state (R)
- the changes in quaternanry strucutre of Hb have dramtic effects on its function as O2 bidning protein
- the sigmoidal O2 binding curve of Hb is diagnostic of cooperative binding which requires >1 ligand binding site and interaction between ligand binding sites
*sigmoidal curve arises due to allosteric effect
2D view of key salt bridges
What is the general mechanism of allosteric proteins
- no ligand: unstable segments (pink) are flexible, green segments are stable in low affinity state
- when ligand binds it stablizes the high affinity conformation of the flexible segment, rest of polypep takes on higher affintiy conformation which is stabilized via protein protein interactions
- when second ligand molecule is bound to second subunit this binding occurs with a higher affinity than binding of first mol
what happend upon bidning of O2 in heme
- binding causes repositioning of the Fe2+ within heme: reducing the pucker of porphyrin ring
- the change in shape of heme pulls the proximal histidine (HF8) causing a shift in position of helix F
*this is one adjustment triggering the T to R transition (includes breaking of ion pairs formed by His HC3)
- look @ diagram and look at new position of His HC3 in R vs T
**IN R STATE (OXY HB) HIS HC3 IS NOT PARTICIPATING IN ION PAIRS THAT STABILIZE THE T STATE (DEOXY HB)
this T -> R transition is induced by O2 binding to Hb
what are ion pairs in deoxy Hb
ion pair between His HC3 of the β subunit and both Asp FG1 of the β subunit and Lys C5 of the α subunit
what does the O2 binding curve look like?
*Sigmoidal*
- in high affinity (R) state in presence of any oxygen
- transition form low to high is the state it is usually in(this is the sigmoidal curve where hemoglobin almsot always resides)
- in lungs: high pO2, Hb binds a lot of oxygen (high affinity state)
- in tissues: low pO2: Hb releases a lot of oxygen (transition)
- the cahnge in theta reflects how much O2 is released to the tissue (about 37% of Hb’s overall capacity)
what is the Hill equation?
* models the occupancy of macromolecules (such as hb)
- for myoglobin which only has 1 binding site the hill plot has constant slope (no allosteric effects, ligand cant influence its own binding)
- hemoglobin has a low and high affinity state, different pressue of O2 will cause diff in the states and how is is binding
*myoglobin has a higher affinity for O2 than hemoglobin
what is the concerted model
- subunits are functionally identical
- subunits can exist in >1 conformation
- all subunits change conformation simultaneously
what sit eh sequential model?
- ligand binding can induce confomrational change in subunits independently
- confomrational change in one subunits promtoes confomrational change in adjacent subunits
- once one changes the ones beside it will be influences, hemoglobin is constantly sshuffeling between these states
Anermia Vs CO poisoning
- Anemia= problems with hemoglobin and its ability to occupy/relase O2
- anemia is a gec in good hb but can still relase the little O2 that they have in tissues
*CO is deadlier than anemia, when binds to hb it takes up binding sites but also induced conformational changes to high affinity state, cannot pick up O2 bc all sites bound
- will not release at tissues because stuck at high affinity
What is the effect of H+ on hemoglobin binding
- does NOT bind to same sites as O2 does
- H+ binds several side chains in Hb, including HC3 (close to carboxy term, 3 aa away)
- protonation of HC3 favours formation of ion pair with ASP FG1 which helps stabilize T state
- as pH drops, adoption of T state enocurages O2 release
WHat are carbamino groups
- co2 comes in and binds to N term, H pops off (this prot then can bind to HIS to further alter salt bridges durther stablizing T state)
- CO2 binding at the amino-termini of Hb forms
carbaminohemoglobin
what of BPG do what is it
2,3 bisphosphoglycerate
- BPG binds in the central cavity of Hb
- It forms salt bridges with the two β subunits
- BPG stabilizes the T-state (deoxy-Hb)
- It therefore reduces the affinity of Hb for O2
This is another example of heterotropic allosteric modulation
*does not interact directly w/ heme
exaplin how BPG alternates with O2 binding
- hemoglobin w/ BPG (only 1 per hemoglobin)
- BPG binds to the T state (deoxy Hb) only
*T state can still bind to O2 just with lower affinity
- Binding of O2 to Hb pushes Hb to the R state, forcing release of BPG
- Conversely, BPG binding in lower O2 environments helps push Hb to release O2
- Hemoglobin in erythrocytes without BPG would barely release any O2 at all in tissues
WHat is the role of BPG in adaption to lower pO2 at high altitude
- the higher you go the lower the oxygen concentration, become anemic
- if you live in higher elevation ur hemoglobin is the same but if you inc concentration of BPG it dec afinity for oxygen at higher altitudes, promote O2 unloading
*without BPG affinity for oxygen is too high and oxygen doesnt get released
what causes sickle cell anemia
- caused by a mutation resulting in an amino acid substitution in Hb
- Hemoglobin A (HbA) vs Hemoglobin D (HbS)
- in HbS, Glu A3 on β-globin (glutemate β6) is changed to valine (point mutation)
- HbS tatramer has 2 fewer negative aminoa cids
- deoxy-HbS tatramers aggregate causing abnormal hydrophobic interaction (water hating so clump togehter to hide from aw env)
- sickle red blood cells contain long HbS fibers
*HbS forms fibers of deoxygenated hemoglobin, makes RBC fragile: capillaries become clogged, fewer RBC, less Hb causing anemia
