Myoglobin and Hemoglobin

Protein Ligand Interactions

reversible, transient process of chemical equilibrium (A+B ←→ AB)

binding molecule (ligand) binds at the binding site via non-covalent forces

equilibrium composition is characterized by Ka = [PL] / [P][L] where large Ka means tight binding; Kd for reverse process

Binding

the fraction of occupied binding sites is theta

[L] is free ligand concentration

Binding Curves

fraction of bound sites depends on the free ligand concentration and Kd (ligand concentration at half saturation when half the binding sites are taken up)

typically ligand concentration is known independent variable

Kd can be found graphically or through least-squares regression

Thermodynamic Connections

interaction strength can be expressed as:

• association constant Ka

• dissociation constant Kd, 1/Ka

• interaction free energy dGnought

strong binding is when Kd < 10 nM

O2 Binding Subunits

each subunit binds one iron containing heme group

alpha subunit has 7 helices; beta has 8

in each subunit the heme group is bound in a hydrophobic pocket between the C, F, and E helices

no covalent attachment between heme and protein

MYOGLOBIN = MONOMER (1 peptide chain)

HEMOGLOBIN = tetramer

Myoglobin

monomeric protein in muscle tissue made of 153 amino acids in a single polypeptide

O2 storage and transport within muscle tissue → increases solubility of O2 in the cells and the diffusion rate within the tissue

binding curve is hyperbolic - at high pO2 it binds oxygen well, at low pO2 it will not release it (bad for transport protein)

Hemoglobin

tetrameric protein (a2b2) tetrameric protein in red blood cells

alpha subunit is 141 amino acids and beta subunit is 143 amino acids

transports O2 to tissues from lungs and CO2 from lungs to tissues

binding curve is sigmoidal

How to Measure O2 Binding

heme group is a strong chromophore that absorbs both in UV and visible range

changes in electronic state of Fe causes changes in absorbance

binding of oxygen can be monitored by UV-Vis spectrophotometry

Why partial pressure of O2?

Mb is used for movement of oxygen within the muscle

Hb is used for moving oxygen from lungs to tissues

pO2 measures amount of O2 dissolved in aqueous solution inside the cell or blood

Myoglobin Function

increase solubility of oxygen

MbO2 diffuses well in water

oxygen storage especially for deep living mammals

bad transport protein

needs tight binding of oxygen in lungs and low binding at tissues - must have multiple binding sites that are able to interact with each other → cooperativity

Positive Cooperativity

first binding event increases affinity at the other binding sites

Hemoglobin Binding Curve

sigmoidal shape because of positive cooperativity from low affinity T state to high affinity R state

Hb has high affinity and low affinity states

at high pO2 it has high affinity for oxygen

at low pO2 it has low affinity for oxygen

T and R States

T (tense) state: Hb conformation in DEOXY-state; low affinity at all 4 binding sites

R (relaxed) state: Hb conformation in the OXY-state; high affinity and binds well at all 4 binding sites - central pocket of molecule is smaller than T state

Hb binds O2 in lungs where pO2 is high and releases in body where pO2 is low - this is why alternation between high and low affinity matters

Effectors of O2 Binding

effector: molecule or ion that binds to a protein or enzyme and changes its activity

• + effectors increase affinity or activity - activator

• - effectors decrease affinity or activity - inhibitor

• homotropic effector binds to same site as substrate (like O2)

• heterotropic effectors bind at a site other than the active site

H+ and CO2 stabilize the T state → decrease O2 affinity by binding to N terminus

BPG stabilizes T state → decrease O2 affinity

The Bohr Effect

increasing acidity (decreasing pH) shifts the Hb binding curve to the right from decreased affinity to O2 → increased O2 delivery to tissues

carbamate formation increases the acidity of the blood → more O2 released stabilizes the T state

2 Bohr protons bind at the C termini of the beta chains and the n termini of the alpha chains in the T state

Haldane Effect

CO2 binding decreases affinity for O2

The BPG Effect

purified Hb behaves differently than Hb in the blood

1. carbamate formation at the N termini of the change

2. H+ affects

3. BPG binding

BPG

bis-phophoglycerate

small triose sugar found in the blood

has 5 negative charges!

1 BPG per tetramer binds to Hb in the central region between the subunits in the T state (pocket too small in the R state) that has 6+ residues → decreases O2 affinity

mutation in BPG binding pocket decreases O2 affinity, e.g. fetal Hb

BPG O2 Binding Curve

decreased affinity and increased p50 with increased [BPG]

has consequences in high altitude and fetal Hb

Effect of BPG at High Altitude

less oxygen available to breathe in from environment at high altitudes

more O2 is dropped off in the body than normal → increased O2 transport than at low [BPG]

p50 is raised as [BPG] increases so it can deliver more oxygen to tissue

Effect of BPG on Fetal Hb

need to get O2 from mom’s blood to fetus

fetal Hb is a mutation that has a stronger affinity for O2 than adult Hb so fetus can get proper oxygen

fetal red blood cells picks up oxygen from the mother’s blood

will gradually be replaced by adult Hb after birth

p50 is lowered as opposed to raised in high altitudes - pulls curve left

Myoglobin

O2 carrying protein in the muscle that primarily facilitates diffusion of O2 in rapidly respiring muscle tissues

protein is 80% alpha-helical with helices A to H having 153 amino acids

structure has 8 alpha helices and 1 heme

heme binding between E and F helicesHE stabilized tertiary structure

hydrophobic interactions, ionic interactions (salt bridges), LDFs stabilize the structure

Heme Binding in Myoglobin

HisF8 coordinates the iron binding, HisE7 restricts the size of the binding site and orientation of oxygen when it binds

H bond prevents superoxide from leaving the heme

Val68 and Phe 43 prevent Fe2+ from being oxidized to Fe3+ so that O2 can bind reversibly

Structural Properties of Hb

• 4 subunits, 2 alpha with 7 alpha helices and 2 beta with 8 alpha helices

• dimer of dimers

• lots of a1b1 contacts (35)

• a1b2 contacts are very important (19)

• not so much b1b2 and a1a2 contact since the protein communicates oxygen binding - needs to talk between subunits

• similar structures between Hb and Mb but only 12% sequence identity

Structural Changes When O2 Binding Hb

changes in heme: Fe2+ gets smaller since e- are being pulled away and moves about 0.6 angstroms into the heme plane

F-helix moves about 1 angstrom (iron pulls on His F8)

changes at a1b2 interface: Asp G1b H bond with Tyr C7a ←→ Asn G4b H bond with Asp G1a

at C termini of alpha and beta chains: O2 stabilizes R state and H bonds stabilizes T conformation

What holds hemoglobin together?

hydrophobic interactions between subunits

interactions between subunits which is important in R to T transition

H bonds and LDFs

Hb Variants

gamma Hb is derived from beta Hb

Fetal Hemoglobin

tetramer with a2y2 composition where gamma subunit is like the beta subunit in adult Hb

lacks His143 b → important for BPG binding (does not bind as tightly as adult form)

• has ser instead which decreases BPG binding since T state stabilizer is gone (removes positive charge so (-) on BPG has less interactions)

higher affinity for O2 at all pO2 values means fetal Hb traps O2 that comes from mother’s Hb

decreased BPG binding biases fetal Hb to R state

Sickle Cell Hb (HbS)

single amino acid change in beta chain (Glu6 changed to Val) makes long fibers in the deoxy T state because pO2 increases

Val6b binds to hydrophobic pocket in a beta chain of another Hb tetramer ONLY in T state

fiber formation makes sickle cell shape in the capillaries so they clog

Hb Hammersmith

PheCD1 (42) b → ser

disrupts hydrophobic heme pocket

Hb Bristol

Val E 11(67) b → Asp

disrupts hydrophobic pocket that holds heme

Hb Svannah

Gly B6(24) b → val

B and E helix interface gets disrupted

Hb Philly

Tyr (35) Phe disrupts H bond at a1b1 interface → destabilizes

HbM

Fe2+ oxidized to Fe3+ so can’t bind O2

aka methemoglobin

Hb Iwate

His F8 (87) a → tyr

disrupts O2 binding site and iron gets oxidized

Hb Milwaukee

Val E11 (67) b → glu

glu side chain stabilizes Fe3+

Hb Boston

His E7 (58) a → tyr

Yakima Mutaiton

Asp99 → His

disrupts H bond that stabilizes T state (now is destabilized)

Kansas Mutation

Asn 102B → Thr

disrupts H bond that stabilizes R state (now is destabilized)

Mutation

in switch region can stabilize T or R state, depends on specific mutation

b1b2 interface will affect BPG binding mostly

mutation near heme binding site will mostly affect O2 binding

mutations that increase O2 affinity decrease cooperativity

mutations that disrupt BPG binding increase O2 affinity